Microbiological Control in Poultry Processing

ABSTRACT

A method of controlling microbial contamination of poultry carcasses in the processing of poultry as food products is described. The method comprises contacting the carcasses with an aqueous medium containing an effective microbial inhibiting amount of active bromine resulting from the addition to the medium of (i) at least one 1,3-dibromo-5,5-dialkylhydantoin in which one of the alkyl groups is a methyl group and the other alkyl group contains in the range of 1 to about 4 carbon atoms or (ii) a solution thereof, or (iii) both of (i) and (ii). Such contacting inhibits contamination of the carcasses by microorganisms, even at least some bacteria that are resistant to antibiotics or antibacterials. Also described are improvements in a poultry chill tank containing an aqueous medium and a plurality of poultry carcasses in contact with the medium. Such improvements result from the presence in the medium of an effective microbial inhibiting amount of active bromine in the medium, which amount results from the addition to water before it enters the tank or while it is in the tank, or both, of (i) at least one of the above 1,3-dibromo-5,5-dialkylhydantoins, and/or a solution thereof.

REFERENCE TO RELATED APPLICATIONS

This is a continuation of commonly-owned copending application Ser. No.11/180,054, filed Jul. 12, 2005, which in turn is a continuation ofcommonly-owned application Ser. No. 10/313,245, filed Dec. 6, 2002, nowU.S. Pat. No. 6,986,910 B2, which in turn is a continuation-in-part ofcommonly-owned application Ser. No. 10/029,329, filed Dec. 21, 2001, nowU.S. Pat. No. 6,908,636 B2, which in turn is a continuation-in-part ofcommonly-owned application Ser. No. 09/893,581, filed Jun. 28, 2001, nowabandoned.

REFERENCE TO OTHER COMMONLY-OWNED APPLICATIONS

Reference is hereby made to the following commonly-owned applications:application Ser. No. 09/088,300, filed Jun. 1, 1998, now U.S. Pat. No.6,068,861 issued May 30, 2000; application Ser. No. 09/296,499, filedApr. 22, 1999, now U.S. Pat. No. 6,110,387 issued Aug. 29, 2000;application Ser. No. 09/323,348, filed Jun. 1, 1999, now U.S. Pat. No.6,303,038 B1 issued Oct. 16, 2001; application Ser. No. 09/404,184,filed Sep. 24, 1999, now U.S. Pat. No. 6,322,822 B1, issued Nov. 27,2001; application Ser. No. 09/442,025, filed Nov. 17, 1999, now U.S.Pat. No. 6,306,441 issued Oct. 23, 2001; application Ser. No.09/451,319, filed Nov. 30, 1999; application Ser. No. 09/451,344, filedNov. 30, 1999, now U.S. Pat. No. 6,352,725 B1, issued Mar. 5, 2002;application Ser. No. 09/456,781, filed Dec. 8, 1999, now U.S. Pat. No.6,495,169 B1, issued Dec. 17, 2002; application Ser. No. 09/483,896,filed Jan. 18, 2000, now U.S. Pat. No. 6,448,410 B1, issued Sep. 10,2002; application Ser. No. 09/484,687, filed Jan. 18, 2000, now U.S.Pat. No. 6,508,954 B1, issued Jan. 21, 2003; application Ser. No.09/484,844, filed Jan. 18, 2000, now U.S. Pat. No. 6,809,205 B1, issuedOct. 26, 2004; application Ser. No. 09/484,891, filed Jan. 18, 2000, nowU.S. Pat. No. 6,495,698 B1, issued Dec. 17, 2002; application Ser. No.09/484,938, filed Jan. 18, 2000, now U.S. Pat. No. 6,565,868 B1, issuedMay 20, 2003; application Ser. No. 09/487,816, filed Jan. 18, 2000, nowU.S. Pat. No. 6,680,070 B1, issued Jan. 20, 2004; application Ser. No.09/506,911, filed Feb. 18, 2000, now U.S. Pat. No. 6,511,682 B1, issuedJan. 28, 2003; application Ser. No. 09/658,839, filed Sep. 8, 2000, nowU.S. Pat. No. 6,375,991 B1, issued Apr. 23, 2002; application Ser. No.09/663,788, filed Sep. 18, 2000, now U.S. Pat. No. 6,348,219 B1, issuedFeb. 19, 2002; application Ser. No. 09/663,948, filed Sep. 18, 2000, nowU.S. Pat. No. 6,299,909 B1 issued Oct. 9, 2001; application Ser. No.09/732,601, filed Dec. 7, 2000, now U.S. Pat. No. 6,506,418 B1, issuedJan. 14, 2003; application Ser. No. 09/775,516, filed Feb. 2, 2001, nowU.S. Pat. No. 6,641,828 B1, issued Nov. 4, 2003; application Ser. No.09/778,228, filed Feb. 6, 2001, now abandoned; application Ser. No.09/785,890, filed Feb. 16, 2001; and application Ser. No. 09/974,622,filed Oct. 9, 2001, now U.S. Pat. No. 6,652,889 B1, issued Nov. 25,2003.

Reference is also hereby made to application Ser. No. 10/028,631, filedDec. 21, 2001, now U.S. Pat. No. 6,919,364 B2.

BACKGROUND

Poultry processing is an area in which microbiological control is ofvital importance. By the very nature of the processing involved thereare numerous opportunities for the poultry to be exposed to variouspathogens in the form of mobile bacteria such as for example Escherichiacoli, Salmonella enteritidis, Salmonella typhimurim, Campylobacterjejuni, Campylobacter coli, Campylobacter lari, and in the form ofbiofilms such as for example Listeria monocytogenes, Pseudomonasfluorescens, Pseudomonas aeruginosa, Enterococcus faecium, andStaphylococcus aureus. The thought of handling, processing and consumingbacteria-infested poultry is revolting in the extreme.

There are several factors which magnify the problem of microbiologicalcontrol in the processing of poultry for use as food. One such factor isthe extremely wide variety of microorganisms that can be encountered insuch processing, and that as reported for example in U.S. Pat. No.6,039,992, sensitivity of a microorganism to a particular antimicrobialagent is not predictive of the sensitivity other microorganisms to thesame agent. Another factor is the ability of various bacterial strainsto develop resistance to antibiotics and antibacterials, such asnalidixic acid, streptomycin, tetracycline, or the like, thereby makingit even harder to discover a way of effectively controlling a broadrange of microorganisms encountered in such processing. Still anotherfactor is the need to effect such control without significantlyaffecting the appearance, texture, quality, and taste of the finishedpoultry products.

Heretofore certain chlorine-based microbiocides have been proposed andused in an attempt to provide suitable sanitation in connection withpoultry processing. Unfortunately while some chlorine-basedmicrobiocides show some effectiveness, they possess a number of seriousshortcomings. For one thing they are not as effective as one might wish.Secondly, they tend to be odorous and in many cases can exert ableaching effect upon the poultry carcasses which can prove unpalatableto the consumer. Moreover, because of the spread of fecal matterassociated with the evisceration of the fowl, fecal bacteria abound.This egregious condition in turn results in high nitrogen levels in thewash waters, and on wet surfaces such as cutting surfaces, conduits,tank surfaces, and other downstream equipment exposed one way or anotherto these wash waters. Unfortunately, the active chlorine species ofcertain chlorine-based microbiocides tend to react with the nitrogenousspecies to form chloroamines which are lachrymators as well as beingcorrosive to metallic surfaces. In fact, as little as 50 ppm of chlorinein aqueous washing tanks containing nitrogenous impurities can producequantities of air-borne lachrymators that are intolerable to plantworkers. Furthermore, the consumption of chlorine values in formingchloramines results in a significant loss of biocidal effectivenessinasmuch as the chloroamines are not biocidally-active species.

Clearly therefore a need exists for a new, more effective, economicallyfeasible way of providing microbiological control in the poultryprocessing industry. Of especial concern, is a need for a way ofeffectively controlling a broad range of microorganisms encountered inthe processing of poultry, and to effectively control contamination ofpoultry carcasses by microorganisms that have developed strains whichare resistant to common antibiotics or antibacterials, such as nalidixicacid, streptomycin, tetracycline, or the like.

BRIEF SUMMARY OF THE INVENTION

This invention fulfills the foregoing need by providing and utilizing incertain highly effective halogen-based microbiocides in the processingof poultry and in the disinfection of equipment, instruments, apparatus,and/or water used in the processing of poultry, and/or of carcassesand/or parts of poultry resulting from the processing of poultry.Microbiocidal agents used pursuant to this invention can be producedeconomically in straightforward processing from relatively low cost rawmaterials and because of their effectiveness, can providemicrobiological control on an economical basis consistent with the needsof the industry.

In particular, this invention provides a method of controlling microbialcontamination of poultry carcasses in the processing of poultry as foodproducts, which method comprises contacting said carcasses with anaqueous medium containing an effective microbial inhibiting amount ofactive bromine resulting from the addition to said medium of (i) atleast one 1,3-dibromo-5,5-dialkylhydantoin in which one of the alkylgroups is a methyl group and the other alkyl group contains in the rangeof 1 to about 4 carbon atoms or (ii) a solution thereof, or (iii) bothof (i) and (ii), said contacting inhibiting contamination of saidcarcasses by microorganisms. Such contacting can be effected in variousways. For example the contacting can be conducted in a chill tankcontaining such active bromine-containing aqueous medium. Alternatively,the contacting can be conducted by spraying, splashing, or pouring theactive bromine-containing aqueous medium onto the carcasses. Thus anyway or combination of ways of bringing about such contact thateffectively controls contamination of the carcasses by microorganismscan be used. 1,3-Dibromo-5,5-dimethylhydantoin is the preferred1,3-dibromo-5,5-dialkylhydantoin for use in conducting this method.

Another embodiment of this invention relates to poultry chill tanks ortheir operation. In a poultry chill tank containing an aqueous mediumand a plurality of poultry carcasses in contact with the aqueous medium,the improvement comprises having an effective microbial inhibitingamount of active bromine present in such medium, such amount of activebromine resulting from the addition to water before it enters the chilltank or while it is in the chill tank, or both, of (i) at least one1,3-dibromo-5,5-dialkylhydantoin in which one of the alkyl groups is amethyl group and the other alkyl group contains in the range of 1 toabout 4 carbon atoms or (ii) a solution thereof, or (iii) both of (i)and (ii), so that contamination of the carcasses by microorganisms isinhibited. Typically, the carcasses are immersed or suspended in theaqueous medium in the tank for a specified period of time, e.g., in therange of about 0.5 to about 2 hours. The presence in the aqueous mediumof the active bromine from the 1,3-dibromo-5,5-dialkylhydantoin providesmicrobiocidal action against a broad range of microorganisms, includingstrains that have developed resistance to other biocides orantibacterials such as nalidixic acid, streptomycin, tetracycline, bothin the chill tank water and on the surfaces of the poultry carcasses incontact with the chill tank water. Thus the contamination of thecarcasses by microorganisms effectively controlled.

In another of its embodiments this invention provides in the processingof poultry, the improvement which comprises disinfecting equipment,instruments, apparatus and/or water used in such processing, and/orcarcasses and/or other parts of poultry resulting from such processing,against contamination by strains of bacteria that have developedresistance to other biocides or antibacterials such as nalidixic acid,streptomycin, and/or tetracycline. Such disinfection is accomplished byuse of a bromine-based microbiocide which is an aqueous microbiocidalsolution of one or more active bromine species resulting from dissolvingin an aqueous medium such as water, a suitable amount of at least one1,3-dibromo-5,5-dialkylhydantoin described above.

The solutions of active bromine used in the practice of the variousembodiments of this invention are derivative products in an aqueousmedium of at least one 1,3-dibromo-5,5-dialkylhydantoin in which one ofthe alkyl groups is a methyl group and the other alkyl group contains inthe range of 1 to about 4 carbon atoms, preferably1,3-dibromo-5,5-dimethylhydantoin. By “derivative product” is meant thatthe active bromine is what forms when the1,3-dibromo-5,5-dialkylhydantoin is dissolved in an aqueous medium suchas water. Such 1,3-dibromo-5,5-dialkylhydantoins are typically availablecommercially in the form of solids. Concentrated aqueous solutions canbe formed from such solids for application with or without furtherdilution to equipment, instruments, or apparatus used in poultryprocessing and added to water used in poultry processing. But forapplication to poultry carcasses or parts thereof, either theconcentrated solution should be further diluted with water before use,or the selected 1,3-dibromo-5,5-dialkylhydantoin solids should be addedto water in proportions yielding the desired microbiocidal dosagedirectly without forming an intermediate more concentrated solution.

In practice, the surfaces to be disinfected are contacted with theaqueous microbiocidal solutions which of course contain amicrobiocidally-effective amount of the microbiocidal agent and/ormicrobiocidal hydrolysis product(s) thereof. Such bromine-basedmicrobiocides are more effective than chlorine-based microbiocidesagainst various bacteria and biofilms. In addition, these bromine-basedmicrobiocides tend to be less odorous than chlorine-based microbiocides,and are essentially devoid of unwanted bleaching activity. Moreover,while some of the bromine-based microbiocides may possibly react withnitrogenous species, such as are present in water and on surfacesassociated with poultry processing, the resultant bromamines would alsopossess microbiological activity. Thus such side reactions would notmaterially decrease the microbiological effectiveness made available tothe poultry processor by use of these bromine-based microbiocides.Furthermore, bromamines generally do not exhibit obnoxious propertiestoward workers in the processing plant whereas chloramines resultingfrom use of certain chlorine-based microbiocides under the sameconditions tend to be powerful lachrymators.

The aqueous microbiocidal solutions used pursuant to the aboveembodiments of this invention can be formed in many cases by adding themicrobiocidal agent itself (i.e., in undiluted form) or as a preformedconcentrated aqueous solution thereof to water being used in one or morepoultry processing operations (e.g., water flowing into chill tanks, orwater already in chill tanks, etc.) to form a diluted microbiocidalsolution of this invention which contacts the surfaces to bedisinfected. Alternatively, a concentrated preformed aqueous solution ofthe microbiocidal agent can be applied directly to the surfaces to bedisinfected (e.g., surfaces of cutting tables, or knives, or etc.), ormore usually such concentrated solution would be mixed with water toform a more dilute solution of the microbiocidal agent which is appliedto the surfaces to be disinfected and/or introduced into water beingused in poultry processing operations. In short, the aqueousmicrobiocidal solutions used pursuant to these embodiments of theinvention can be made in whole or in part from water already in use orto be used in the poultry processing operations, or can be made entirelyfrom water separate from that used or to be used in the poultryprocessing. In each such case, the contacting of the aqueousmicrobiocidal solution however produced and/or applied to the surfacesresults in effective disinfection.

Various embodiments and features of this invention will be still furtherapparent from the ensuing description and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical depiction of the effect of chill tankmicrobiocidal treatments on growth of Pseudomonas species on chickenskin.

FIG. 2 is a graphical depiction of the effect of chill tankmicrobiocidal treatments on growth of total aerobic bacteria on chickenskin.

FIG. 3 is a graphical depiction of the effect of chill tankmicrobiocidal treatments on growth of Pseudomonas species on chickenskin.

FIG. 4 is a graphical depiction of the effect of chill tankmicrobiocidal treatments on growth of total aerobic bacteria on chickenskin.

FIG. 5 is a graphical depiction of the results obtained in testsinvolving use of bromine species derived from1,3-dibromo-5,5-dimethylhydantoin in eradicating HPC (heterotrophicplate count) bacteria in a biofilm at concentrations in water of 0.5 and5 ppm as bromine.

FIG. 6 is a graphical depiction of the results obtained in testsinvolving use of bromine species derived from1,3-dibromo-5,5-dimethylhydantoin in eradicating planktonic HPC(heterotrophic plate count) bacteria at concentrations in water of 0.5and 5 ppm as bromine.

FURTHER DETAILED DESCRIPTION OF THE INVENTION

The bromine-based microbiocides for use in disinfection of equipment,instruments, apparatus, and/or water used in the processing of poultry,and/or of carcasses and/or parts of poultry resulting from theprocessing of poultry pursuant to this invention is an aqueousmicrobiocidal solution of one or more active bromine species. Thesespecies result from dissolving in water at least one1,3-dibromo-5,5-dialkylhydantoin in which one of the alkyl groups is amethyl group and the other alkyl group contains in the range of 1 toabout 4 carbon atoms. Thus these preferred biocides comprise1,3-dibromo-5,5-dimethylhydantoin,1,3-dibromo-5-ethyl-5-methylhydantoin,1,3-dibromo-5-n-propyl-5-methylhydantoin,1,3-dibromo-5-isopropyl-5-methylhydantoin,1,3-dibromo-5-n-butyl-5-methylhydantoin,1,3-dibromo-5-isobutyl-5-methylhydantoin,1,3-dibromo-5-sec-butyl-5-methylhydantoin,1,3-dibromo-5-tert-butyl-5-methylhydantoin, and mixtures of any two ormore of them. Of these biocidal agents,1,3-dibromo-5-isobutyl-5-methylhydantoin,1,3-dibromo-5-n-propyl-5-methylhydantoin, and1,3-dibromo-5-ethyl-5-methylhydantoin are, respectively, preferred, morepreferred, and even more preferred members of this group from the costeffectiveness standpoint. Of the mixtures of the foregoing biocides thatcan be used pursuant to this invention, it is preferred to use1,3-dibromo-5,5-dimethylhydantoin as one of the components, with amixture of 1,3-dibromo-5,5-dimethylhydantoin and1,3-dibromo-5-ethyl-5-methylhydantoin being particularly preferred. Themost preferred member of this group of microbiocides is1,3-dibromo-5,5-dimethylhydantoin. This compound is available in themarketplace in tablet or granular form under the trade designationsAlbrom™ 100T biocide, Albrom™ 100 PF and Albrom™ 100PC biocide(Albemarle Corporation).

When a mixture of two or more of the foregoing1,3-dibromo-5,5-dialkylhydantoin biocides is used pursuant to thisinvention, the individual biocides of the mixture can be in anyproportions relative to each other.

Methods for producing 1,3-dibromo-5,5-dialkylhydantoins are known andreported in the literature.

If desired, the 1,3-dibromo-5,5-dialkylhydantoins can be dissolved in asuitable innocuous, harmless, water-soluble organic solvent with orwithout water to form a solution which can be applied to surfaces ofequipment, instruments, or apparatus. Depending upon the solvent used,the surfaces can then be further washed with clean water to removeresidues from such solvent. Besides increasing the amount of1,3-dibromo-5,5-dialkylhydantoin that can be put into solution thusfacilitating formation of a concentrated solution, e.g., on the premisesof the poultry processing, such a concentrated solution when dilutedsuch as by addition to process water being used on the premisespossesses microbiocidal activity from the1,3-dibromo-5,5-dialkylhydantoin. Thus aqueous solutions used pursuantto this invention can contain suitably small amounts of an innocuous,harmless, water-soluble organic solvent, which non-toxic, at least atthe dosage levels involved, such as acetonitrile.

The amount (concentration) of the selected microbiocide utilized in thepractice of this invention will vary depending on various factors suchas the particular 1,3-dibromo-5,5-dialkylhydantoin being used, thenature and frequency of prior microbiocidal treatments, the types andnature of the microorganisms present, the amount and types of nutrientsavailable to the microorganisms, the nature and extent of cleansingactions, if any, taken in conjunction with the microbiocidal treatment,the surface or locus of the microorganisms being treated, and so on. Inany event, a microbiocidally-effective amount of the diluted aqueoussolution of the microbiocide of this invention will be applied to orcontacted with the microorganisms. Typically the diluted solution willcontain a microbiocidally-effective amount of active bromine in therange of about 2 to about 1000 ppm (wt/wt), preferably in the range ofabout 2 to about 500 ppm (wt/wt), and more preferably in the range ofabout 25 to about 250 ppm (wt/wt), active bromine being determinable byuse of the conventional DPD test procedure. A particularly preferredrange for the 1,3-dibromo-5,5-dialkylhydantoins used in ordinarysituations (e.g., washing hard surfaces such as tables, walls, floors,conveyor machinery or parts thereof such as conveyor belts or shackles,and knives or cutting blades) is in the range of about 50 to about 150ppm (wt/wt) of active bromine. When contacting poultry carcasses oredible parts thereof with aqueous solutions formed from at least one1,3-dibromo-5,5-dialkylhydantoin, it is especially preferred to use inthe water for washing or otherwise contacting the poultry carcasses oredible parts thereof, a microbiocidally effective amount of activebromine that does not significantly or appreciably bleach the skin ofthe carcass or have a significant or appreciable adverse effect upon theorganoleptic taste of cooked meat from the poultry such as the breastmeat and thigh meat. Such amount is typically within the range of about0.5 to about 100 ppm (wt/wt), and preferably in the range of about 5 toabout 100 ppm (wt/wt) of active bromine as determinable by the DPD testprocedure. When contacting poultry carcasses with the biocidal aqueousmedium in a chill tank or by spraying, splashing, or pouring thebiocidal aqueous medium onto the poultry carcasses, under conditionswhere strains of bacteria may be present having increased resistance toantibiotics or antibacterials such as, for example, nalidixic acid,streptomycin, tetracycline, or the like, the amount of active brominederived from the 1,3-dibromo-5,5-dialkylhydantoin is desirably in therange of about 20 or 30 to about 100 ppm (wt/wt). For example, incarefully controlled tests highly effective results against a variety ofbacterial species have been achieved with active bromine concentrationsderived from 1,3-dibromo-5,5-dimethylhydantoin ranging from about 34 ppm(wt/wt) to about 78 ppm (wt/wt), including tests conducted at about 56ppm (wt/wt) of active bromine. It will be understood that departuresfrom the foregoing ranges can be made whenever deemed necessary ordesirable, and such departures are within the spirit and scope of thisinvention.

Depending upon the way in which the microbiocide of this invention isbeing used, a microbiocidally-effective amount of the microbiocides ofthis invention can extend from as little as about 2 ppm up to as high asthe maximum water solubility of the particular1,3-dibromo-5,5-dialkylhydantoin microbiocidal agent being used, at thetemperature at which such microbiocidal agent is being used.

There are two different types of procedures that can be used fordetermining active bromine content. For measuring concentrations in thevicinity of above about, say, 500 ppm or so (wt/wt) of active bromine,starch-iodine titration is the preferred procedure. On the other hand,where concentrations are below this approximate level, the conventionalDPD test procedure is more suitable, as this test is designed formeasuring very low active halogen concentrations, e.g., active bromineconcentrations in the range of from zero to about 5 ppm (wt/wt). Infact, where the actual concentration of active bromine is between, say,about 5 ppm and about 1100 ppm (wt/wt), the test sample is typicallydiluted with pure water to reduce the actual concentration to be in therange of about 2 to about 5 ppm of active bromine before making the DPDanalysis. It can be seen therefore that while there is no criticalhard-and-fast concentration dividing line between which procedure touse, the approximate values given above represent a practicalapproximate dividing line, since the amounts of water dilution of moreconcentrated solutions when using the DPD test procedure increase withincreasing initial active halogen concentration, and such largedilutions can readily be avoided by use of starch-iodine titration whenanalyzing the more concentrated solutions. In short, with suitablydilute solutions use of the DPD test procedure is recommended, and withmore concentrated solutions use of starch-iodine titration isrecommended.

The starch-iodine titration procedure for determination of activehalogen has long been known. For example, chapter XIV of Willard-Furman,Elementary Quantitative Analysis, Third Edition, D. Van NostrandCompany, Inc., New York, Copyright 1933, 1935, 1940 provides adescription of starch-iodine titration. While details of standardquantitative analytical procedures for determination of active halogenin such product solutions by starch-iodine titration may vary from caseto case, the results are normally sufficiently uniform from one standardprocedure to another as not to raise any question of unreliability ofthe results. A recommended starch-iodine titration procedure is asfollows: A magnetic stirrer and 50 milliliters of glacial acetic acidare placed in an iodine flask. The sample (usually about 0.2-0.5 g) forwhich the active halogen is to be determined is weighed and added to theflask containing the acetic acid. Water (50 milliliters) and aqueouspotassium iodide (15%, wt/wt; 25 milliliters) are then added to theflask. The flask is stoppered using a water seal. The solution is thenstirred for fifteen minutes, after which the flask is unstoppered andthe stopper and seal area are rinsed into the flask with water. Anautomatic buret (Metrohm Limited) is filled with 0.1 normal sodiumthiosulfate. The solution in the iodine flask is titrated with the 0.1normal sodium thiosulfate; when a faint yellow color is observed, onemilliliter of a 1 wt % starch solution in water is added, changing thecolor of the solution in the flask from faint yellow to blue. Titrationwith sodium thiosulfate continues until the blue color disappears. Theamount of active halogen is calculated using the weight of the sampleand the volume of sodium thiosulfate solution titrated. In this way, theamount of active bromine in an aqueous product solution, regardless ofactual chemical form, can be quantitatively determined.

The standard DPD test for determination of low levels of active bromineis based on classical test procedures devised by Palin in 1974. See A.T. Palin, “Analytical Control of Water Disinfection With SpecialReference to Differential DPD Methods For Chlorine, Chlorine Dioxide,Bromine, Iodine and Ozone”, J. Inst. Water Eng., 1974, 28, 139. Whilethere are various modernized versions of the Palin procedures, therecommended version of the test is fully described in Hach WaterAnalysis Handbook, 3rd edition, copyright 1997. The procedure for “totalchlorine” (i.e., active chlorine) is identified in that publication asMethod 8167 appearing on page 379, Briefly, the “total chlorine” testinvolves introducing to the dilute water sample containing activehalogen, a powder comprising DPD indicator powder, (i.e.,N,N′-diethyldiphenylenediamine), KI, and a buffer. The active halogenspecies present react(s) with KI to yield iodine species which turn theDPD indicator to red/pink. The intensity of the coloration depends uponthe concentration of “total chlorine” species (i.e., active chlorine”)present in the sample. This intensity is measured by a calorimetercalibrated to transform the intensity reading into a “total chlorine”value in terms of mg/L Cl₂. If the active halogen present is activebromine, the result in terms of mg/L Cl₂ is multiplied by 2.25 toexpress the result in terms of mg/L Br₂ of active bromine.

In greater detail, the DPD test procedure is as follows:

-   1. To determine the amount of species present in the water which    respond to the “total chlorine” test, the water sample should be    analyzed within a few minutes of being taken, and preferably    immediately upon being taken.-   2. Hach Method 8167 for testing the amount of species present in the    water sample which respond to the “total chlorine” test involves use    of the Hach Model DR 2010 calorimeter. The stored program number for    chlorine determinations is recalled by keying in “80” on the    keyboard, followed by setting the absorbance wavelength to 530 nm by    rotating the dial on the side of the instrument. Two identical    sample cells are filled to the 10 mL mark with the water under    investigation. One of the cells is arbitrarily chosen to be the    blank. To the second cell, the contents of a DPD Total Chlorine    Powder Pillow are added. This is shaken for 10-20 seconds to mix, as    the development of a pink-red color indicates the presence of    species in the water which respond positively to the DPD “total    chlorine” test reagent. On the keypad, the SHIFT TIMER keys are    depressed to commence a three minute reaction time. After three    minutes the instrument beeps to signal the reaction is complete.    Using the 10 mL cell riser, the blank sample cell is admitted to the    sample compartment of the Hach Model DR 2010, and the shield is    closed to prevent stray light effects. Then the ZERO key is    depressed. After a few seconds, the display registers 0.00 mg/L Cl₂.    Then, the blank sample cell used to zero the instrument is removed    from the cell compartment of the Hach Model DR 2010 and replaced    with the test sample to which the DPD “total chlorine” test reagent    was added. The light shield is then closed as was done for the    blank, and the READ key is depressed. The result, in mg/L Cl₂ is    shown on the display within a few seconds. This is the “total    chlorine” level of the water sample under investigation.

In the practice of this invention the microbiocidal system can be usedin various ways. For example, a microbiocidally effective amount of amicrobiocide of this invention is applied to the locus of themicroorganisms to be eradicated or controlled so that the microbiocidalsystem comes in contact with these microorganisms. The application canbe made by thorough application by pouring, spraying, wet mopping,flooding, and/or wet wiping infested or potentially infested surfaces orareas of the processing equipment and environs such as flooring, walls,tables, conveyors, stanchions, conduits, tanks, and drains with abiocidally-effective amount of an aqueous solution of the microbiocide.Where applicable and possible, portions of the processing apparatus canbe immersed in an aqueous solution of the microbiocide, with temporarydisassembly, if necessary. Such applications should be conductedroutinely on a frequency sufficient to ensure that exposure of thepoultry being processed to dangerous microorganisms, such as bacteriaand biofilms is prevented to the greatest extent possible. For bestresults these operations should be conducted in conjunction orassociation with thorough cleaning operations such as scrubbing,scouring, scraping and, otherwise removing infestations of biofouling orbiofilms, whether visible or invisible. After contacting themicroorganisms with the microbiocide for a suitable period of time toensure penetration into polysaccharide slimes and other defensemechanisms of various species of these microorganisms, the entiredisinfected area should be washed, e.g., hosed down, with clean waterand preferably the washings themselves should be disinfected withadditional microbiocide of this invention before discharge. The contacttimes will of course vary depending upon the frequency and thoroughnessof the cleaning and disinfection operations and the identity andconcentration of the particular microbiocidal solution being employed.Generally speaking contact times may fall in the range of from about afew minutes to a few hours, but any period of time that effects theeradication or control of the microbial population in the poultryprocessing areas should be used and is within the scope of thisinvention.

Another mode of applying the microbiocidally-effective amounts ofsolid-state microbiocides of these embodiments of the invention is tocause the microbiocide to be leached into water streams passing throughconduits and into tanks or other washing devices utilized in theprocessing of the poultry. For example, suitable solid forms of themicrobiocide such as tablets, briquettes, pellets, nuggets, or granulesare placed in suitable feeding devices through which a stream of wateris passed. The passage of the water through the bed of the microbiocideresults in the stream continuously dissolving small quantities of themicrobiocide to thereby provide microbiocidally effective amounts of themicrobiocide in the water. 1,3-Dibromo-5,5-dimethylhydantoin isespecially preferred for use in this mode of application because of itsrelatively low solubility and thus relatively slow rate of dissolutionin water at ambient room temperatures. This translates into relativelylong periods of use before need of refilling the device holding thesolids. By way of example, the solubility of1,3-dibromo-5,5-dimethylhydantoin in water at 75° F. (ca. 24° C.) is 405ppm expressed as Cl₂ whereas the solubilities ofN,N′-bromochloro-5,5-dimethylhydantoin and of the commercial mixture ofN,N′-bromochloro-5,5-dimethylhydantoin and1,3-dichloro-5-ethyl-5-methylhydantoin at the same temperature are,respectively, 890 ppm and 1905 ppm, both expressed as Cl₂.

An especially cost-effective, operationally efficient, and highlypreferred way of forming aqueous microbiocidal solutions of one or more1,3-dibromo-5,5-dialkylhydantoins in which one of the alkyl groups is amethyl group and the other alkyl group contains in the range of 1 toabout 4 carbon atoms, most preferably 1,3-dibromo-5,5-dimethylhydantoin,(“dibromodialkylhydantoin(s)”) comprises passing water through a bed ofone or more such dibromodialkylhydantoin(s) in granular, nugget, pellet,tablet or other non-powdery particulate form (“bed”) disposed in acanister, tank, or other similar vessel (“tank”). Preferably the tankhas a pressure sealable port at its upper portion for periodicallyreplenishing the contents of the bed, and the water is caused to flowupwardly through a portion of the bed. More preferably, the tank iselongated in an upward direction so that the bed is longer from top tobottom than from side to side, this upward water flow is dispensed intothe bed to flow upwardly through only a lower portion of the bed, andthence substantially horizontally through a port disposed between thelower and the upper portions of the bed and tank. In this way the upperportion of the bed serves as a reserve supply of contents of the bedwhich automatically feeds into the lower portion of the bed undergravity as the lower portion of the bed is slowly but substantiallyuniformly dissolved away in the water flow. Thus in this operation thewater flow is preferably at least a substantially continuous flow, andmost preferably, is a continuous flow. Methods for producing granules,tablets or other non-powdery particulate forms of1,3-dibromo-5,5-dimethylhydantoin are described in detail incommonly-owned copending applications PCT/US01/01541, 01/01545, and01/01585, all filed Jan. 17, 2001, each claiming priority based onrespective earlier-filed corresponding U.S. applications. Excellentprocess technology for producing 1,3-dibromo-5,5-dimethylhydantoin foruse in making such granules, tablets or other non-powdery particulateforms is described in detail in commonly-owned copending applicationPCT/US 01/01544, filed Jan. 17, 2001, claiming priority based on anearlier-filed corresponding U.S. application. The disclosures of eachsuch PCT and U.S. application is incorporated herein by reference.Particularly preferred apparatus for use in conjunction with suchgranules, tablets or other non-powdery particulate forms of thesedibromodialkylhydantoin(s) in forming aqueous microbiocidal solutionsthereof is available from Neptune Chemical Pump Company, a division ofR.A. Industries, Inc., Lansdale, Pa. 19446, as “Bromine Feeders” ModelsBT-15, BT-40, BT-42, BT-80, BT-160, BT-270, and BT-350, or equivalent.Excellent results are achieved using combinations of Model BT-40 withgranules or nuggets of 1,3-dibromo-5,5-dimethylhydantoin Albrom™ 100PCbiocide available from Albemarle Corporation. Single charges of suchmicrobiocides in tablet or granular form in such device can providecontinuous highly-effective microbiocidal activity in bodies of end usewater at ordinary outdoor temperatures for as long as five (5) monthswithout need for replenishment.

Another suitable method of effecting contact between the microbiocideand the microorganisms is to pump an aqueous solution containing amicrobiocidally-effective amount of the microbiocide through theconduits and into the tanks or other washing devices, such as scaldingtanks and chill tanks, utilized in the processing of the poultry.Variants of this procedure include dispensing portion-wise as by gravitydripping an aqueous solution of the microbiocide directly into a tank orother vessel in which poultry are to be or are being processed.

A further mode of application pursuant to these embodiments of theinvention involves applying to or contacting the poultry itself,typically promptly before and preferably after slaughter anddefeathering, with an aqueous solution of the microbiocide. Afterproviding a suitable contact time to eradicate bacteria on the surfacesof the poultry, the poultry can then be washed down to remove both theexcess microbiocide and the dispatched microbial population from theexposed surfaces of the fowl itself. The internal organs of the fowlafter slaughter can also be treated and washed down in the same manner.The application(s) of the microbiocidal solution(s) in this manner cantake any suitable form, e.g., use of aqueous sprays containing amicrobiocidally-effective amount of the microbiocide being used, orimmersion of the fowl or internal organs thereof in one or more tankscontaining aqueous solutions of microbiocidally-effective amounts of themicrobiocide being used.

Preferably two or more of the foregoing methods of application of themicrobiocides of this invention are used. Thus in a preferred embodimenta microbiocide of these embodiments of the invention is applied by (i)periodically contacting at least portions, if not all, of the poultryprocessing apparatus to disinfection or sanitization with amicrobiocidally-effective amount of an aqueous solution of at least oneof the above 1,3-dibromo-5,5-dialkylhydantoins, and (ii) contacting theexposed surfaces of the poultry with a microbiocidally-effective amountof an aqueous solution of at least one of the above1,3-dibromo-5,5-dialkylhydantoins, before and/or after, preferablyafter, dispatching the fowl, and most preferably after defeathering thefowl. In another preferred embodiment, a microbiocide of theseembodiments of the invention is applied by (i) periodically contactingat least portions, if not all, of the poultry processing apparatus todisinfection or sanitization with a microbiocidally-effective amount ofan aqueous solution of at least one the above1,3-dibromodialkylhydantoins, and (ii) contacting the edible portionsand/or internal organs of the dispatched fowl with amicrobiocidally-effective amount of an aqueous solution of at least oneof the above 1,3-dibromo-5,5-dialkylhydantoins.

Particularly preferred processes of this invention are those wherein thefowl are processed by a series of steps which comprise the following:(a) suspending the fowl in moving clamps or shackles, (b) stunning, butnot killing, the fowl such as by use of a suitable gas, or by contactingat least the heads of the fowl with a water-applied electric shock tostun the fowl, e.g., by immersing the heads in a water bath carrying asuitable current to effect the stunning, (c) cutting the jugular veinsand/or carotid arteries at the neck of the stunned fowl either manuallywith a knife or automatically with a mechanical cutting device, (d)draining blood from the carcasses, (e) scalding the birds with hotwater, e.g., in a scalding tank, to facilitate feather removal, (f)defeathering the fowl, (g) removing the heads and feet from the fowl,(h) eviscerating the fowl either manually with a knife, or automaticallywith mechanical evisceration apparatus, (i) separating the viscera fromthe carcasses, (j) washing the carcasses, and (k) chilling thecarcasses, e.g., in water such as by passage of the carcasses through atleast one and often two chill tanks, or by air chilling. The scaldingstep will typically be conducted at water temperatures in the range ofabout 50 to about 60° C., with the lower temperatures being preferredfor retention of normal yellow-colored skin. The higher temperatureswill more usually be used in connection with turkeys and spent egg-layerhens. The chilling temperatures used will typically reduce the carcasstemperature to below about 4° C., with final temperatures of thefinished carcasses for shipment being as low as about −2° C. Other stepscan be included and in some cases one or more of the steps (a) through(j) may be altered or revised or the sequence of the steps may to someextent be altered or revised, to adapt to given circumstances. Examplesof extra steps that may be included are inspection steps, e.g., bygovernmental regulatory personnel, and wax-dipping in the case of waterfowl to enhance the extent of defeathering. Inspections are oftenconducted subsequent to the evisceration step, such as before separatingthe viscera from the carcasses. Wax dipping will typically be used whenprocessing waterfowl, the feathers of which typically are more difficultto remove than, say, chickens. Wax dipping will typically be performeddirectly after use of feather-picking machines which utilize rubber“fingers” to beat off the feathers. The wax dipping step will typicallyinvolve dipping the partially defeathered carcass into a molten waxcontained in a tank, allowing the wax to harden on the carcass, and thenremoving the wax coating as by peeling it off along with feathersembedded in the wax. This operation can be repeated as desired, beforeproceeding to the next step in the process, e.g., removal of the headsand feet. One illustrative example of a suitable revision of thesequence of steps, would be to conduct step (g) before step (d) insteadof after step (f). Upon a reading of this disclosure, other suitablesequence revisions may well become obvious to one of ordinary skill inthe art, and thus need not be further elaborated upon here.

In the above processing, the microbiocidal action of the1,3-dibromo-5,5-dialkylhydantoin microbiocides of the invention can beapplied at any of a variety of suitable stages in the operation. Forexample. an applicable microbiocidal solution of this invention can beapplied to any or all of the processing equipment used including knives,conveying apparatus, the surfaces of emptied scaling tanks, defeatheringapparatus, (e.g., rubber “fingers” etc.), knives and mechanicalapparatus used for cutting or eviscerating the fowl, all surfaces thatcome in contact with the blood or the viscera of the fowl, includingtables, conveyor belts, etc., and all surfaces that come in contact withthe carcasses after separation of the viscera therefrom. The applicablesanitizing solutions of this invention can be applied to by immersion,spraying, flooding, or any other way of ensuring that themicrobiocidally-effective solution contacts the surfaces that contain orare exposed to the undesirable microorganisms such as bacteria and/orbiofilm (biofouling).

Another way by which, in the above processing, the microbiocidal actionof the microbiocides of this invention can be applied involves includinga microbiocidally-effective amount of the microbiocide to the waterbeing used at one or more stages of the processing. Thus the water inthe scalding tank(s) and/or in the chill tank(s) can be so treated.Another mode is to include a microbiocidally-effective amount of themicrobiocide to the water used in washing the carcasses and the visceraat various points where these parts are handled, separated, and/orprocessed. The dosage levels at these different points in the processingcan be the same or different as deemed necessary or desirable.

The practice and advantages of this invention are illustrated by thefollowing non-limiting Examples.

EXAMPLE 1

A study was conducted to determine the effectiveness of1,3-dibromo-5,5-dimethylhydantoin (DBDMH) as a disinfectant, when usedin the poultry chill tank for the control of carcass bacteria. The studyincluded, for purposes of comparison, use of sodium hypochlorite as adisinfectant under the same conditions. In this study the bacterialspecies used were genetically marked strains resistant to several commonantibiotics and antibacterials. Four treatment groups as identified inTable 1 were used in this study. TABLE 1 Treatment Br₂/Cl₂ Target Group¹Treatment^(2,3,5) Concentration 1 Non-disinfected unchilled control NoneNo disinfectant added Carcasses spotted with 10⁹ bacteria Carcassesdried and immediately rinsed 2 Non-disinfected chilled control None Nodisinfectant added to chill tank Carcasses spotted with 10⁹ bacteriaCarcasses dried, chilled, and rinsed 3 Sodium hypochlorite treatment⁴⁴15 ppm Cl₂ 15 ppm Cl2 equivalent added to chill tank (+/−20%) Carcassesspotted with 10⁹ bacteria Carcasses dried, chilled and rinsed 4 DBDMHtreatment 34 ppm Br₂ 34 ppm Br₂ added to chill tank (+/−20%) (equivalentto 15 ppm Cl₂) Carcasses spotted with 10⁹ bacteria Carcasses dried,chilled and rinsed¹Four (4) different treatments were administered to six (6) blocks withfive (5) carcasses per block.²Chlorine/Bromine determinations confirmed zero added levels forTreatment Group 2. Chlorine levels were confirmed for Treatment Group 3and Bromine levels were confirmed for Treatment Group 4.³1:1 mixture of genetically marked strains of Salmonella Agona andSalmonella Kentucky was used for all carcass bacteria spotting.⁴Sodium hypochlorite solution (CAS No. 7681-52-9, Aldrich No. 42,504-4).⁵Carcasses were immersed in the chill tank for 80 +/− 5 min. (TreatmentGroups 2, 3, and 4).

The test procedures used were as follows:

A) Randomization and Blinding

Randomization was accomplished by employing a complete block design fortreatments groups 2, 3, and 4 using computer-generated randomizednumbers. The trial was blinded to the technicians performing bacteriapreparation and enumeration via color-coding of the chill tanks.Blinding was accomplished by having one person placing test materialsinto the chill tank and other technicians performing all other duties.Trial number, chill tank number, color code and block number identifiedeach chill tank. Carcasses were double wingbanded for identification andto eliminate any possibility of bias from any of the three chill tanktreatments. Stomacher bags and sample bottles for whole-bird rinse werepre-labeled to include block number, color code, and carcass number.Agar plates were labeled block number, carcass number, color code, anddilution rate.

B) Source, Preparation, and Enumeration of Bacteria

1) Bacterial Source: The bacterial stock cultures used in this studywere two genetically marked strains (Salmonella Agona (FDA code FMK14O1]and Salmonella Kentucky [FDA code FMK1402]) obtained from the FDA/CFSANlaboratory. Both were modified for resistance to nalidixic acid,streptomycin, and tetracycline. They were stored at approximately 4-8°C. In addition, the isolates are being maintained long term as describedin the Federal Register, Volume 61, No. 144, Jul. 25, 1996, page 38924.

2) Bacterial Culture Purification: The stock cultures were transferredseparately onto Nutrient Agar media supplemented with approximately 30ug/mL of each antibiotic (e.g., nalidixic acid, streptomycin andtetracycline) and incubated at 37+/−2° C. for 24+/−2 hours. Cultureswere removed from agar surfaces with five-mL phosphate buffer dilutionwater and centrifuged in sterile centrifuge tubes approximately twominutes to settle agar particles. The supernatants were transferred tosterile centrifuge tubes and centrifuged again to obtain completeseparation of cells. Fresh Nutrient Agar whole plates (supplemented withantibiotics) were swabbed from the supernatant using sterile swabs.Plates were incubated at 37+/−2° C. for 24+/−2 hours. This process wasrepeated over two additional days to ensure pure, viable strains.

3) Preparation of Stationary Growth Phase Cultures: From thepurification process above, the bacterial cultures were inoculated intoseparate antibiotic supplemented Nutrient Broth media and incubated at37+/−2° C. until the stationary growth phase was reached. Establishedgrowth curves have shown this to be approximately 18 hours (opticaldensity >1000 Formazin Turbidity Units or FTU). Optical density readingswere taken at 17 hours and cultures were used at 18 hours.

4) Preparation of Bacterial Stock Solution for Spotting: Each brothculture from 3) was diluted as necessary to achieve a target of at least10⁹ bacteria per mL. An equal (1:1) mixture of these two dilutions wasprepared and used to spot carcasses. Both dilutions and the mixture wereenumerated as per 6) below.

5) Spotting Time: Bacteria were spotted onto the carcasses within 30min. after removal from the incubator.

6) Culture Plating and Enumeration: The dilution, plating and countingprocedures described in “Standard Methods for the Examination of Waterand Wastewater,” 20th Edition, Section 921 SC were followed. Exceptionswere as follows: (i) All water samples were refrigerated atapproximately 4-8° C. and plated within 48 hours of collection. (ii) Theplating volumes were 1 mL on whole plates (1:1 bacterial mixture, andbacterial dilutions from 4) above and chill tank water dilutions), and0.5 mL on half plates (carcass rinse water dilutions). (iii) Theinoculum was distributed across the surface of the agar by rotating thepetri dish by hand. (iv) Incubation temperature and time was 37+/−2° C.for 24+/−2 hours. (v) Plates were placed in the incubator upright forone hour for drying purposes, then turned upside down for the remainingincubation time. (vi) Counting was conducted manually, without an aid.

C) Carcass Source and Preparation

1) Carcasses were purchased at a local retail store. After removing thegiblets, carcass weights were approximately 3.0-5.0 lbs (1361-2268 g).This weight range is within industry standards of processed weightscommonly found in retail stores and reflect a normal population ofchicken carcasses.

2) Upon collection, all carcasses were immediately placed in a coolerwithout ice and immediately transported approximately 20 miles to thetesting laboratory. At the test facility, carcasses were refrigerated at4-8° C.

3) Carcasses were removed from the refrigerator within 4-6 hr prior tospotting with bacteria. They were drained in a wire basket forapproximately five (5) min., wingbanded for identification, and weighedwithin 15 min. of bacterial spotting.

D) Carcass Bacteria Spotting

1) Carcasses were placed flat, on an open, covered laboratory bench andspotted as follows: (i) Using the bacterial mixture prepared in B) 4)above, all carcasses within the block were spotted externally along eachbreast feather track (7 per track) and legs (3 per leg) with twenty-50microliter aliquots (1 mL total). (ii) After bacteria application,carcasses were allowed to dry at ambient temperature for 25-35 min. Thisdrying period represented time from defeathering and evisceration, andallowed time for the bacteria to adhere to the skin. (iii) The surfacesspotted did not touch any object prior to chill tank immersion.

E) Disinfectant Preparation and Measurement

1) Stock Solution Preparation: Stock solutions were prepared withinthree hr of Time 0 (carcass immersion). (i) To prepare the DBDMH stocksolution, ten grams of DBDMH was added to each one (1) liter of sterilewater and mechanically stirred for at least 20 min. The stock solutionwas passed through a 200-mesh screen and filtered through two coursefilters (Fisher 09-790-14F, course porosity, fast flow rate, pleated).The DBDMH Stock solution was diluted as necessary and Br₂ levelsdetermined in triplicate, as described in E) 2) below. (ii) To preparethe sodium hypochlorite stock solution, concentrated sodiumhypochlorite, commercial grade, was obtained from Aldrich and used asreceived. Fifteen mL sodium hypochlorite stock solution was diluted tothree liters and Cl₂ levels determined in triplicate, as described in E)2) below.

2) Bromine/Chlorine Determination: A Hach Pocket Colorimeter Test Kitfor Bromine (Hach Item Number 4670001) was employed to determinebromine/chlorine concentrations.

3) Disinfectant Addition And Br₂/Cl₂ Determination: The disinfectantswere added to the chill tank just prior to ice addition. Enoughdisinfectant was added to account for the dilution from the ice. Thetargeted Cl₂ and Br₂ concentrations were 25 ppm (+/−20%) and 56 ppm(+/−20%) respectively, prior to ice addition. Target concentrationsafter ice addition, were 15 ppm Cl₂ and 34 ppm Br₂ and were calculatedbased on the measured values prior to ice addition.

F) Chill Tank Preparation and Chilling Procedures

1) Experimental Unit: Experimental chill tanks (28-gallon plasticcontainers) were used to simulate commercial chilling techniques. Eachexperimental unit contained 40 liters total volume and provided 8 litersof water per carcass.

2) Ingredients: Each chill tank contained the following: TABLE 2 OrderAdded Ingredient Added Chill Tank Ingredient per 40,000 mL Chill TankWater Cumulative 1 Water Added 22,000 mL 22,000 mL 2 DBDMH or SodiumDisinfectant stock solution plus enough sterile water to 24,000 mLHypochlorite stock equal 2000 mL solutions 3 Carcasses added 5 carcassesspotted with 10⁹ bacteria N/A 4 Ice 16,000 grams (one gram equalsapproximately one mL) 40,000 mLTOTAL 40,000 mL chill tank water plus disinfectant

3) Chilling Procedure: A block of three chill tanks were run at onetime. Each chill tank was initiated at least 20 min. apart to allowappropriate time for post-chill data collection. Processing roomtemperature was recorded within 15 min. of carcass immersion.

4) Carcass Temperature Determination: Carcass external skin temperaturewas determined along the breast feather track prior to carcass immersionand after 80+/−5 min. chilling time.

5) Control Treatment Carcasses Rinsed: Carcasses in treatment group 1(non-disinfected, unchilled control) were rinsed 25-35 min. afterspotting, using the “whole bird” rinse procedure described in G) below.

6) Bromine/Chlorine Determination: The bromine or chlorine level, asapplicable, for each chill tank (treatment groups 2, 3, and 4), weredetermined prior to carcass immersion, 45+/−5 min., 60+/−5 min., andafter 80+/−5 min. chilling time.

7) Carcass Immersion (Time Zero) and Ice Addition: Five, previouslyspotted carcasses, were completely immersed in the chill tank water. Icewas added within 5+/−2 min. of carcass immersion to lower carcasstemperature. The water temperature was maintained at 4.5° C. (40° F.USDA HACCP minimum rule) or less.

8) Chill Tank Water Temperature Determination: The water temperature wasdetermined as soon as possible after time zero and at 20+/−2 min.thereafter.

9) At time zero and each 5 min. period thereafter, the carcasses werelifted twice by completely lifting from the chill tank mixture forapproximately 5 seconds assuring that each carcass cavity was drainedeach time.

10) Chill Tank Water Samples: After 45+/−5 min. and 60+/−5 min. carcassexposure time, a 5-mL water sample was removed from each chill tank forbacterial enumeration as per B) 6) above. These samples representedshorter chill times that may be experienced in the processing industry.

11) Chilling Time: Total carcass exposure in the chill tank was 80+/−5min.

12) After carcass removal from the chill tanks, the following eventsoccurred: (i) External carcass temperature was measured along the breastfeather track, (ii) All chilled carcasses underwent the “whole bird”rinse procedure described below, (iii) A 5-mL water sample was taken forSalmonella bacteria counts. To each water sample, sodium sulfite (0.10mL of 1000 ppm solution) was added immediately to neutralize the effectof DBDMH and sodium hypochlorite, (iv) Carcasses were weighed andpercent moisture uptake was determined, (v) Residual chill tank waterBr₂/Cl₂ levels were determined, as applicable. The control treatment(containing no disinfectants) residual values were recorded as Cl₂, (vi)Chill tank room temperature was measured within 20 min. after trialcompletion, (vii) A water sample was taken.

G) Whole Bird Rinse Procedures

1) Prior to rinsing carcasses, sodium sulfite (8 mL of 1000 ppmsolution) was equally added to the 400 mL Butterfield's phosphatediluent (BPD) solution to neutralize the effect of DBDMH and sodiumhypochlorite.

2) Carcasses were taken out of the chill tanks at the end of the 80+/−5min. testing period. Carcass temperature was taken, and each was allowedto drain for approximately one (1) minute. Each individual carcass wasaseptically transferred to a sterile stomacher bag.

3) 400 mL BPD was poured directly to the internal cavity of each carcassin the sterile stomacher bag. The remaining “whole bird” rinse techniquewas followed as described in the Federal Register Vol. 61, No. 144, p.38921.

4) The rinse solutions were transferred from each stomacher bag intosample bottles (completely labeled), and refrigerated at approximately4-8° C.

The water used in this study was from a deep-water well, previouslycertified by Maryland Health Department. It had not been chlorinatedsince at least September, 2000. All water pH, chlorine/bromine levels,and bacteria content (Salmonella, Escherichia coli and Coliform) weredetermined on water used in this trial. For all parameters measured,Statgraphics (ver. 6.1) Multifactoral Analysis of Variance procedure wasused to compare means of the treatment groups. Significant differencesbetween the means were identified by the Least Significant Differencetest and presented in the tables. Significant differences at the p<0.05level were reported.

The results of this study as regards bacteria reduction are summarizedin Tables 3 and 4. Table 3 compares the results achieved in terms ofreductions from the non-chilled control treatment group. Table 4compares the results achieved in terms of reductions from the chilledcontrol treatment group. TABLE 3 Reduction Log₁₀ Percent from controlreduction reduction Difference non-chilled DBDMH NaOCl DBDMH NaOCl Log₁₀Carcass 7.466 4.963 99.9999% 99.9985% +2.503 bacteria reduction Totalbacteria 5.557 4.342 99.9995% 99.9888% +1.215 reduction

TABLE 4 Log₁₀ Percent Reduction from reduction reduction Differencecontrol chilled DBDMH NaOCl DBDMH NaOCl Log₁₀ 45 min. water 4.266 3.35699.9867% 99.8805% +0.910 bacteria reduction 60 min. water 4.513 3.45199.9969% 99.9108% +1.062 bacteria reduction 80 min. water 4.632 3.68899.9972% 99.9390% +0.944 bacteria reduction Carcass 6.643 4.140 99.9994%99.9910% +2.503 bacteria reduction Total bacteria 5.029 3.813 99.9985%99.9663% +1.216 reduction

As seen from the data in Tables 3 and 4, although NaOCl was effective inthe reduction of bacterial contamination, DBDMH was significantly moreeffective in this regard. More particularly:

1) Based on the data generated in this study,1,3-dibromo-5,5-dimethylhydantoin (DBDMH) is an effective disinfectantwhen used in the poultry chill tank. The numbers of Salmonella bacteriawere significantly reduced both in the chill tank water and on thepoultry carcasses in the presence of DBDMH when compared to the controltreatments.

2) The log₁₀ reductions of DBDMH were significantly greater than NaOClin the carcass rinse, chill tank water (with the exception of the80-minute reduction) and combined carcass and chill tank water.

3) At the three chill times tested, bacterial reduction was demonstratedusing DBDMH. There was approximately a one log difference in bacterialreduction between DBDMH and NaOCl at all chill times.

4) Adverse carcass effects were found. However, these effects wereunchanged from prechill to post-chill processing with the use of eitherNaOCl or DBDMH.

5) After the 80 minute chilling period, disinfectant residual levels, asapplicable, were detected across all blocks. This indicates that initialdisinfectant levels were enough to provide for bacterial reductionthroughout the chilling process.

6) At the concentration employed in this study (34 ppm Br₂) andcomparing to the control chill tank treatment: (i) DBDMH reduced chilltank water Salmonella spp. by 4.3 4.6 log₁₀ bacteria reduction, (ii)DBDMH reduced carcass Salmonella spp. by 6.6 log₁₀ bacteria reduction,(iii) DBDMH effectively reduced Salmonella spp. when used as adisinfectant in the chill tank, (iv) DBDMH did not adversely affectpoultry carcass quality, (v) DBDMH was effective across a range of chilltimes (45-80 minutes) that may be used by the poultry industry, (vi) Ingeneral, DBDMH was at least comparable to or in most cases, a moreeffective chill tank disinfectant than NaOCl, regardless of chillingtime.

EXAMPLE 2

Comparative tests were conducted to determine the effect on poultrycarcass bacteria (Escherichia coli field strain) during a normal1.5-hour chill tank immersion in water containing differentmicrobiocidal compositions. The effect of these treatments on theresidual chill tank water was also investigated. Carcasses were firstimmersed in a warm bath containing 10⁴ E coli per mL of liquid.Carcasses were then immersed in chill tanks containing normal organicfluids (blood, fat, skin, and meat particles) and containing one of therespective microbiocidal compositions under test. Total bacteria countof whole bird (both inside and outside) was used to determine efficacyof various microbiocidal compositions. The microbiocidal compositionstested were sodium hypochlorite (Clorox® bleach), the combination ofsodium hypochlorite and sodium bromide, and1,3-dibromo-5,5-dimethylhydantoin, the first two materials being forcomparative purposes. In this group of tests, 100 birds were used andthe chill water was composed per liter of 950 mL of water, 50 mL ofblood, 10 g of ground abdominal fat, 10 g of meat particles, and 10 g ofskin with fat.

The experimental design used in this group of tests is summarized inTable 5. TABLE 5 Test Active Ingredient Group or equivalent TestMaterial Disinfectant Level 1 None No disinfectant¹ 2 Chlorine (50 ppm)Clorox ® bleach 12.5% Cl₂ (dilution 1:2,500), Contains 50 ppm chlorine²3 Chlorine (100 ppm) Clorox ® bleach 12.5% Cl₂ (dilution 1:1,250)Contains 100 ppm chlorine 4 Chlorine (150 ppm) Clorox ® bleach 12.5% Cl₂(dilution 1:800) Contains 150 ppm chlorine 5 Chlorine Bleach and LiquidSodium Bromide (1:1 mole ratio mix) (50 ppm total) Bleach dilution1:3,500 & NaBr dilution 1:28,000 Contains 50 ppm chlorine equivalent(1:1 Cl₂ equivalent) 6 Chlorine Bleach and Liquid Sodium Bromide (1:1mole ratio mix) (100 ppm total) Bleach dilution 1:1,750 & NaBr dilution1:14,000 Contains 100 ppm chlorine equivalent (1:1 Cl₂ equivalent) 7Chlorine Bleach and Liquid Sodium Bromide (1:1 mole ratio mix) (150 ppmtotal) Bleach dilution 1:1,200 & NaBr dilution 1:9,300 Contains 150 ppmchlorine equivalent (1:1 Cl₂ equivalent) 8 Chlorine DBDMH (equivalent to50 ppm Cl₂ level)-0.9 g per liter (50 ppm total) Contains 50 ppmchlorine equivalent (1:1 Cl₂ equivalent) 9 Chlorine DBDMH (equivalent to100 ppm Cl₂ level)-1.7 g per liter (100 ppm) Contains 100 ppm chlorineequivalent (1:1 Cl₂ equivalent) 10 Chlorine DBDMH (equivalent to 150 ppmCl₂ level)-3.4 g per liter (150 ppm) Contains 150 ppm chlorineequivalent (1:1 Cl₂ equivalent)¹Negative control contained contaminated (bacteria 2.67 × 10⁵ per mL)water.²Positive control is normal poultry industry practice of adding 50 ppmchlorine.

The microbiocidal solution of this invention was prepared in thefollowing manner:

-   1. To form a stock solution, 100 g of    1,3-dibromo-5,5-dimethylhydantoin (DBDMH) was stirred into 10 liters    (10,000 mL) of water for 20 minutes. After filtration, the resulting    clear solution contains 1300 mg per liter as Br₂. This corresponds    to 580 mg per liter (or 580 ppm Cl₂) when expressed as Cl₂.-   2. The washing solution of DBDMH having a content of 50 ppm of Cl₂    equivalent solution was formed by mixing 875 mL of the above stock    solution with 10 liters (10,000 mL) of the above prepared chicken    chill water solution. The washing solutions of DBDMH containing 100    ppm Cl₂ equivalent and 150 ppm Cl₂ equivalent were prepared in the    same manner except that 1750 mL and 2625 mL, respectively, of the    above stock solution were mixed with separate 10-liter portions of    the above prepared chicken chill water solution.

Table 6 summarizes the results obtained in this group of tests. TABLE 6Carcass Bacteria Reduction Test Test Material Whole Bird Bacteria MeanChill Water Group Disinfectant Level Reduction (%) Bacteria Reduction(%)¹ 1 No disinfectant Control² Control 2 Clorox ® bleach³, 50 ppm Cl₂  6.6%   8.2% 3 Clorox ® bleach, 100 pp, Cl₂   28.2%   32.8% 4 Clorox ®bleach 150 ppm Cl₂   41.1%   59.3% 5 NaBr 50 ppm Cl₂ equivalent + Bleach  14.8%   18.4% 6 NaBr 100 ppm Cl₂ equivalent + Bleach   38.5%   41.6% 7NaBr 150 ppm Cl₂ equivalent + Bleach   73.5%   84.7% 8 DBDMH, 50 ppm Cl₂equivalent 99.9999% 99.9999% 9 DBDMH, 100 ppm Cl₂ equivalent 99.9999%99.9999% 10 DBDMH, 150 ppm Cl₂ equivalent 99.9999% 99.9999%¹The value represents bacteria count per mL of treatment water.²Negative control contained contaminated (bacteria 2.67 × 10⁵ per mL)water.³Positive control is normal poultry industry practice of adding 50 ppmchlorine.

EXAMPLE 3

This group of tests was conducted to determine the effect of Clorox®bleach, Aquatize® biocide, and 1,3-dibromo-5,5-dimethylhydantoin (DBDMH)on carcass bacteria (Escherichia coli field strain) residual after1.5-hour in a chill tank “soup”. Tests were conducted with soups at pH7, pH 8 and pH 9 (adjusted by trisodium phosphate) for whole birdbacteria counts. Tests at pH 8 were conducted for individual bacteriacounts.

In general the tests involved normal processing of 56-day-old birds andimmersing the carcasses first in a warm bath containing 10⁴ per mLEscherichia coli, 10⁴ per mL Salmonella enteritidis, 10⁴ per mLPseudomonas aeruginosa, 10⁴ per mL Campylobacter jejuni, and 10⁴ per mLspoilage bacteria each from three strains (Listeria monocytogenes andShigella sonnei). The carcasses were then immersed in a chill tank“soup”, containing normal organic fluids (blood, fat, skin, and meatparticles) and containing the microbiocides on the test.

Tables 7 and 8 summarize the experimental design of these group oftests. TABLE 7 Whole Bird Bacteria Counts at pH 7, pH 8, and pH 9 Birds/Test Group Test Material (Chill Tank) Reps Rep 1 None (Control) 5 10 2Clorox ® Bleach (20 ppm Cl₂ equivalent) 5 10 3 Aquatize ® biocide (1:500dilution) 5 10 4 Aquatize ® biocide (1:1000 dilution) 5 10 5 DBDMH (10ppm Cl₂ equivalent) 5 10 6 DBDMH (20 ppm Cl₂ equivalent) 5 10

TABLE 8 Individual Bird Bacteria Counts at pH 8 Birds/ Test Group TestMaterial (Chill Tank) Reps Rep 7 None (Control) 5 5 8 Clorox ® Bleach(20 ppm Cl₂ equivalent) 5 5 9 DBDMH (10 ppm Cl₂ equivalent) 5 5 10 DBDMH(20 ppm Cl₂ equivalent) 5 5

The bacteria stock solution used for this group of tests was prepared bygrowing each bacteria sample in the appropriate broth shown in Table 9.Each such broth had a volume of at least 500 mL and the bacteria wereallowed to grow for at least 6 hours. The containers were observed andnot allowed to develop a heavy, cloudy visual appearance which wouldindicate that the growth had developed for too long a period. Thus thesolutions had the appearance of only being foggy or somewhat unclear.TABLE 9 Broth Treatments Organism¹ Broth Plating Media S. sonneiNutrient Broth Nutrient Agar L. Brain Heart Infusion Broth Brain HeartInfusion Agar monocytogenes E. coli Brain Heart Infusion Broth BrainHeart Infusion Agar S. enteritidis Tryptic Soy Broth Tryptic Soy Agar P.aeruginosa Tryptic Soy Broth Tryptic Soy Agar C. jejuni Brucella BrothBrucella Agar¹ Shigella sonnei, Listeria monocytogenes, Escherichia coli, Salmonellaenteritidis, Pseudomonas aeruginosa, and Campylobacter jejuni.

The microbiocidal solution of this invention was prepared in thefollowing manner:

-   1. To form a stock solution, 100 g of    1,3-dibromo-5,5-dimethylhydantoin (DBDMH) was stirred into 10 liters    (10,000 mL) of water for 20 minutes. After filtration, the resulting    clear solution contains 1300 mg per liter as Br₂. This corresponds    to 580 mg per liter (or 580 ppm Cl₂) when expressed as Cl₂.-   2. The chill water solution of DBDMH having a content of 10 ppm of    Cl₂ equivalent was formed by mixing 175 mL of the above stock    solution with 10 liters (10,000 mL) of the above prepared chicken    chill water solution. The chill water solution of DBDMH containing    20 ppm Cl₂ equivalent and 150 ppm Cl₂ equivalent were prepared in    the same manner except that 350 mL of the above stock solution were    mixed with another 10-liter portion of the above prepared chicken    chill water solution.

Table 10 shows the composition of the “chicken soup” used in thesetests. TABLE 10 Composition of “Chicken Soup” Material¹ Material per2100 mL² Water Added 1840 mL Bacteria Stock Solution 200 mL Blood 40 mLChicken Abdominal Fat (ground) 30 g Thigh Meat Particles 30 g Chickenskin with fat 10 g TOTAL 2100 mL equivalent¹The combined material was chilled overnight.²The material was ground and aggressively stirred prior to use.

The procedure used for whole bird wash sampling was as follows:

-   1. All samples were kept at <50 degrees Fahrenheit following    collection.-   2. Microbiological analyses of samples began within 24 hours of    sample collection.-   3. Information on the individual sample identification, date of    collection, time of collection (phase during shift), treatment group    and location of sample point were recorded on each sample bottle.-   4. At each defined sample time, carcasses were taken individually    from the processing line wearing latex or rubber gloves. The gloves    were rinsed with alcohol between each collection.-   5. Any excess fluid was drained off from the carcass. Each    individual carcass was transferred to a sterile stomacher bag.-   6. To each carcass contained in the sterile stomacher bag, 400 mL of    Butterfield's Phosphate Diluent (BPD) was added while making sure to    pour the BPD into the inside of the carcass cavity. The carcass was    rinsed inside and out with a rocking motion for one minute (ca. 35    RPM). This was best done by grasping the broiler carcass with one    hand and the closed top of the bag with the other then rocking with    a reciprocal motion in a 18-24 inch arc, assuring that all surfaces    (interior and exterior of the carcass) were rinsed.-   7. The rinse solutions from each stomacher bag was transferred into    the sample bottles, taking care to ensure that the information on    the date of collection, time of collection (phase during shift),    treatment group and location of sampler point matched that of the    sample.-   8. Each bottle was sealed with parafilm and placed into a styrene    container with crushed or dry ice or frozen freezer packs for    overnight delivery to a testing laboratory.-   9. All filled styrene containers were held in a chilled (not below    freezing) area until within 1 to 2 hours of courier collection for    shipment.

Quantitative or qualitative determinations for bacterial organisms wereconducted according to the following methodologies:

Aerobic plate counts—Counting rules according to BAM 8th ed., Chapter 3.

Coliform and E. coli counts—AOAC, 991.14, Petrifilm.

Salmonella—AOAC 986.35, ELISA presumptive screen.

Salmonella—USDA LC-75, incidence.

Campylobacter—USDA LC-69, incidence.

Listeria—USDA LC-57, incidence.

In greater detail the trial events and experimental design used in thisgroup of tests were as follows:

-   a) Test microorganisms used were:

Escherichia coli ATCC 11229

Pseudomonas aeruginosa ATCC 15442

Salmonella enteritidis ATCC 13076

Shigella sonnei ATCC 9290

Listeria monocytogenes ATCC 7644

Campylobacter jejuni ATCC 29428

-   b) Test Procedure: All test strains were grown individually at    35° C. for 24 hours in the media specified in Table 12. Cells were    harvested by centrifugation at 10,000×g for 10 minutes and washed    twice with Butterfield's Phosphate Buffer (BPB of pH 7.2). Cells    were resuspended in BPB to obtain a cell suspension of approximately    1.0×10⁸ CFU/mL for each microorganism. The target inoculum levels    were approximately 10⁶ CFU/mL in the final test solutions. In the    cases of S. enteritidis and P. aeruginosa the species were washed by    pouring into prepared sterile centrifuge tubes with cheesecloth    filters. The culture was then pelleted and washed using above    techniques and repeated 3 times.-   c) The birds (56 days old) were processed under normal commercial    conditions.-   d) The bacteria were added to a large batch of the “chicken soup”,    and then aliquots of the resultant mixture were distributed equally    among the chill waters used for each test. Then the particular    disinfectant composition under test was added to one of the chill    waters. The chill waters each contained 10⁴ per mL Escherichia coli,    10⁴ per mL Salmonella enteritidis, 10⁴ per mL Campylobacter jejuni,    and 10⁴ per mL spoilage bacteria each from three strains (Listeria    monocytogenes, Pseudomonas aeruginosa, and Shigella sonnei).-   e) Birds were added to each of ten 50-gallon containers containing    these respective treatments (or control) and were kept in the    containers for the 1.5 hour chilling period.-   f) During the 1.5 hour chilling period, the contents were vigorously    stirred every 10 minutes.-   g) After the 1.5 hour chilling period, the whole birds were placed    in individual sterile stomacher bags and the whole bird rinse (as    described above) was conducted and samples of the rinse were placed    on the appropriate agar plates. The plates were placed in the    incubator for 24 hours at 37° C. Then the plates were read after 24    hours to determine total count on each plate.

The results of this group of tests are summarized in Tables 11 and 12.TABLE 11 Whole Bird Total Aerobic Bacteria (% Reduction)¹ WaterTreatment Water pH 7 Water pH 8 Water pH 9 None (Control) — — — Clorox ®Bleach 15% 15%  2% (20 ppm Cl₂ equivalent) Aquatize ® biocide 76% 71%64% (1:500 dilution) Aquatize ® biocide 42% 45% 33% (1:1000 dilution)DBDMH (10 ppm 85% 82% 78% Cl₂ equivalent) DBDMH (20 ppm 99% 98% 96% Cl₂equivalent)¹Each value represents 50 birds per treatment.

TABLE 12 Disinfecting Treatment (average bacteria count per bird)^(2,3)Clorox Bleach DBDMH DBDMH Organism¹ Control (20 ppm) (10 ppm) (20 ppm)S. sonnei 4551 3552 456 12 L. monocytogenes 2463 2065 262 6 E. coli 30552759 357 4 S. enteritidis 3969 3160 560 10 P. aeruginosa 2783 2280 289 9C. jejuni 1282  981 183 15 Mean % Reduction — 18.3% 85.8% 98.8% FromControl¹ Escherichia coli, Salmonella enteritidis, Pseudomonas aeruginosa,Campylobacter jejuni, listeria monocytogenes, and Shigella sonnei²NOTE: Cross contamination is more likely in a processing environmentwhere birds were processed and samples taken for individual culturedetermination.³Each value represents 25 birds per treatment.

EXAMPLE 4

A study was conducted to determine the effect of Clorox® bleach, and1,3-dibromo-5,5-dimethylhydantoin (DBDMH) on carcass bacteria residualafter 1.5 hour in a chill tank solution and spoilage 20-day shelf lifelongevity (caused by bacteria contamination). Tests were conducted at pH8 (adjusted by trisodium phosphate). Skin pigmentation (Minolta ColorMeter L value or Lightness, a value or redness and b value oryellowness) were determined before and post-processing.

In general the study involved normal processing of 56-day-old birds,immersing carcasses first in a warm bath containing 10⁴ per mLEscherichia coli, 10⁴ per mL Salmonella enteritidis, 10⁴ per mLPseudomonas aeruginosa, 10⁴ per mL Campylobacter jejuni, and 10⁴ per mLspoilage bacteria each from three strains (Listeria monocytogenes andShigella sonnei). Carcass were then immersed in a chill tank “soup”,containing normal organic fluids (blood, fat, skin, and meat particles)and containing various disinfectants (termed test materials).

Four test groups of birds were tested at pH 8 for whole bird bacteriacounts. Table 13 sets forth the experimental design for these wholebacteria count tests. TABLE 13 Test Group Test Material (Chill Tank)Reps Birds/Rep 1 None (Control) 6 10 2 Clorox ® bleach (20 ppm Cl₂equivalent) 6 10 3 DBDMH (10 ppm Cl₂ equivalent) 6 10 4 DBDMH (20 ppmCl₂ equivalent) 6 10

A DBDMH stock solution and test solutions, a bacteria stock solution,and a “chicken soup” were prepared as in Example 3. In addition, thebacterial broth treatments, the whole bird wash sampling procedure, andthe methodologies used for quantitative or qualitative determinationsfor bacterial organisms were conducted as in Example 3.

In greater detail the trial events and experimental design used in thisgroup of tests were as follows:

-   a) Test microorganisms used were:

Escherichia coli ATCC 11229

Pseudomonas aeruginosa ATCC 15442

Salmonella enteritidis ATCC 13076

Shigella sonnei ATCC 9290

Listeria monocytogenes ATCC 7644

Campylobacter jejuni ATCC 29428

-   b) Test Procedure: All test strains were grown individually at    35° C. for 24 hours in the media specified in Table 12. Cells were    harvested by centrifugation at 10,000×g for 10 minutes and washed    twice with Butterfield's Phosphate Buffer (BPB of pH 7.2). Cells    were resuspended in BPB to obtain a cell suspension of approximately    1.0×10⁸ CFU/mL for each microorganism. The target inoculum levels    were approximately 10⁶ CFU/mL in the final test solutions. In the    cases of S. enteritidis and P. aeruginosa the species were washed by    pouring into prepared sterile centrifuge tubes with cheesecloth    filters. The culture was then pelleted and washed using above    techniques and repeated 3 times.-   c) The birds (56 days old) were processed under normal commercial    conditions.-   d) The bacteria were added to a large batch of the “chicken soup”,    and then aliquots of the resultant mixture were distributed equally    among the chill waters used for each test. Then the particular    disinfectant composition under test was added to one of the chill    waters. The chill waters each contained 10⁴ per mL Escherichia coli,    10⁴ per mL Salmonella enteritidis, 10⁴ per mL Campylobacter jejuni,    and 10⁴ per mL spoilage bacteria each from three strains (Listeria    monocytogenes, Pseudomonas aeruginosa, and Shigella sonnei).-   e) Birds were added to each of ten 50-gallon containers containing    these respective treatments (or control) and were kept in the    containers for the 1.5 hour chilling period.-   f) During the 1.5 hour chilling period, the contents were vigorously    stirred every 10 minutes.-   g) After the 1.5 hour chilling period, the whole birds were placed    in a commercial refrigerator for 20-days of storage.-   h) Skin pigmentation (using Minolta Color Meter L or Lightness, a or    redness and b or yellowness) were determined on all birds before and    immediately after post-processing chilling.-   i) For Day 0, a total of 5 whole birds per treatment were randomly    chosen from each treatment and placed in individual sterile    stomacher bag and the whole bird rinse (as described in Example 3)    was carried out and samples of the rinse were placed on appropriate    agar plates.-   j) For each of succeeding days 2, 4, 6, 8, 10, 12, 14, 16, 18, and    20, a total of 5 whole birds per treatment were randomly chosen from    each treatment and placed in individual sterile stomacher bags and    the whole bird rinse (as described in Example 3) was conducted and    samples of the rinse were placed on the appropriate agar plates.-   k) All of the treated agar plates were placed in an incubator for 24    hours at 35° C. Plates were read after 24 hours to determine total    count on each plate.

The results of these tests are summarized in Tables 14-27. TABLE 14Percentage of Total Bacteria Reduction From Control (Dayspost-processing) Water Day Day Day Day Day Day Treatment Day 0 Day 2 Day4 Day 6 Day 8 10 12 14 16 18 20 None — — — — — — — — — — — (Control)Clorox ® Bleach 22.5 23.1 22.2 25.2 26.0 25.7 25.9 26.5 23.2 23.12 20.5(20 ppm) DBDMH 77.8 77.3 76.8 77.1 74.6 71.9 69.2 66.2 61.9 58.5 53.7(10 ppm) DBDMH 99.5 99.4 99.2 98.5 97.3 95.1 91.2 84.3 71.2 68.0 67.2(20 ppm)

TABLE 15 Average skin TBA Values¹ (Days post-processing) Water TreatmentDay 0 Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Day 14 Day 16 Day 18 Day 20None 0.14a 0.31a 0.45a 0.69a 0.88a 1.23a 1.36a 1.66a 2.08a 2.39a 3.02a(Control) Clorox ® Bleach 0.10a 0.42a 0.68a 0.72a 0.90a 1.10a 1.49a1.73a 2.19a 2.51a 2.88a (20 ppm) DBDMH 0.20a 0.54a 0.79a 0.54a 0.76a1.20a 1.77a 1.94a 2.33a 2.45a 2.92a (10 ppm) DBDMH 0.22a 0.36a 0.46a0.71a 0.75a 1.22a 1.53a 1.87a 2.19a 2.68a 2.73a (20 ppm)¹NOTE: Means within a row without a common superscript are significantlydifferent (P < 0.05) as determined by Least Significant Difference.

TABLE 16 Skin Pigmentation Value (Minolta Color Meter)¹ Mean Pre-ChillMean Post-Chill Minolta Value Minolta Value Water Treatment L a b L a bNone (Control) 62.84a 5.32a 15.42a 58.84a 5.93a 16.84a Clorox ® Bleach63.62a 5.49a 15.94a 58.84a 5.64a 16.16a (20 ppm) DBDMH (10 ppm) 61.55a5.14a 15.63a 58.84a 6.09a 16.22a DBDMH (20 ppm) 60.77a 5.69a 15.67a58.84a 6.24a 16.37a¹NOTE: Means within a row without a common superscript are significantlydifferent (P < 0.05) as determined by Least Significant Difference.

TABLE 17 Effect of Disinfection Treatment on Day 0 DisinfectingTreatment (average bacteria count per bird) Clorox bleach DBDMH DBDMHOrganism Control (20 ppm) (10 ppm) (20 ppm) S. sonnei 3687 2948 845 8 L.monocytogenes 2569 2281 528 13 E. coli 3879 2310 861 22 S. enteritidis1678 1064 292 12 P. aeruginosa 2974 2681 743 6 C. jejuni 2276 1935 51917 Mean % Reduction — 22.5% 77.8% 99.5% From Control

TABLE 18 Effect of Disinfection Treatment on Day 2 DisinfectingTreatment (average bacteria count per bird) Clorox DBDMH DBDMH OrganismControl (20 ppm) (10 ppm) (20 ppm) S. sonnei 4119 3241 962 12 L.monocytogenes 2749 2442 601 19 E. coli 4193 2604 966 31 S. enteritidis1921 1191 344 18 P. aeruginosa 3313 2889 820 9 C. jejuni 2534 2114 57325 Mean % Reduction — 23.1% 77.3% 99.4% From Control

TABLE 19 Effect of Disinfection Treatment on Day 4 DisinfectingTreatment (average bacteria count per bird)² Clorox DBDMH DBDMHOrganism¹ Control (20 ppm) (10 ppm) (20 ppm) S. sonnei 4664 3528 1101 19L. monocytogenes 2920 2751 670 28 E. coli 4379 3001 1050 49 S.enteritidis 2152 1309 394 27 P. aeruginosa 3592 3127 931 13 C. jejuni2830 2267 627 39 Mean % Reduction From — 22.2% 76.8% 99.2% Control

TABLE 20 Effect of Disinfection Treatment on Day 6 DisinfectingTreatment (average bacteria count per bird)² Clorox DBDMH DBDMHOrganism¹ Control (20 ppm) (10 ppm) (20 ppm) S. sonnei 5424 3802 1288 37L. monocytogenes 3176 3071 741 55 E. coli 4769 3142 1124 100 S.enteritidis 2426 1347 433 55 P. aeruginosa 4141 3454 1013 25 C. jejuni3113 2423 671 78 Mean % Reduction From — 25.2% 77.1% 98.5% Control

TABLE 21 Effect of Disinfection Treatment on Day 8 DisinfectingTreatment (average bacteria count per bird)² Clorox DBDMH DBDMHOrganism¹ Control (20 ppm) (10 ppm) (20 ppm) S. sonnei 5969 4008 1604 76L. monocytogenes 3407 3474 880 107 E. coli 5194 3438 1364 204 S.enteritidis 2764 1519 507 104 P. aeruginosa 4768 3798 1268 48 C. jejuni3353 2594 834 157 Mean % Reduction From — 26.0% 74.6% 97.3% Control

TABLE 22 Effect of Disinfection Treatment on Day 10 DisinfectingTreatment (average bacteria count per bird)² Clorox DBDMH DBDMHOrganism¹ Control (20 ppm) (10 ppm) (20 ppm) S. sonnei 6292 4415 1954156 L. monocytogenes 3854 3767 1096 218 E. coli 5683 3694 1621 401 S.enteritidis 3116 1605 616 212 P. aeruginosa 5243 4305 1485 91 C. jejuni3589 2844 1043 294 Mean % Reduction From — 25.7% 71.9% 95.1% Control

TABLE 23 Effect of Disinfection Treatment on Day 12 DisinfectingTreatment (average bacteria count per bird)² Clorox DBDMH DBDMHOrganism¹ Control (20 ppm) (10 ppm) (20 ppm) S. sonnei 6890 5030 2347323 L. monocytogenes 4348 4195 1335 442 E. coli 6316 3902 2063 775 S.enteritidis 3461 1819 740 413 P. aeruginosa 5743 4720 1730 186 C. jejuni4133 3213 1309 594 Mean % Reduction From — 25.9% 69.2% 91.2% Control

TABLE 24 Effect of Disinfection Treatment on Day 14 DisinfectingTreatment (average bacteria count per bird)² Clorox DBDMH DBDMHOrganism¹ Control (20 ppm) (10 ppm) (20 ppm) S. sonnei 7768 5313 2848657 L. monocytogenes 4781 4755 1564 843 E. coli 6762 4279 2581 1453 S.enteritidis 3901 2055 919 832 P. aeruginosa 6426 5200 2055 363 C. jejuni4454 3446 1551 1191 Mean % Reduction From — 26.5% 66.2% 84.3% Control

TABLE 25 Effect of Disinfection Treatment on Day 16 DisinfectingTreatment (average bacteria count per bird)² Clorox DBDMH DBDMHOrganism¹ Control (20 ppm) (10 ppm) (20 ppm) S. sonnei 7970 6108 35131286 L. monocytogenes 5263 5228 1901 1646 E. coli 7201 4692 3005 2933 S.enteritidis 4281 2328 1081 1711 P. aeruginosa 6969 6005 2560  700 C.jejuni 4898 3733 1880 2259 Mean % Reduction From — 23.2% 61.9% 71.2%Control

TABLE 26 Effect of Disinfection Treatment on Day 18 DisinfectingTreatment (average bacteria count per bird)^(2,3) Clorox DBDMH DBDMHOrganism¹ Control (20 ppm) (10 ppm) (20 ppm) S. sonnei 9004 6957 42421604 L. monocytogenes 5799 5694 2221 1985 E. coli 7725 5097 3617 3645 S.enteritidis 4835 2613 1286 2074 P. aeruginosa 7814 6869 3087  826 C.jejuni 5319 3900 2359 2835 Mean % Reduction From — 23.1% 58.5% 68.0%Control

TABLE 27 Effect of Disinfection Treatment on Day 20 DisinfectingTreatment (average bacteria count per bird)^(2,3) Clorox DBDMH DBDMHOrganism¹ Control (20 ppm) (10 ppm) (20 ppm) S. sonnei 9288 7409 49411834 L. monocytogenes 6419 6506 2678 2238 E. coli 8272 5635 4460 4036 S.enteritidis 5335 2976 1513 2258 P. aeruginosa 8604 7886 3853  908 C.jejuni 5789 4332 2789 3059 Mean % Reduction From — 20.5% 53.7% 67.2%Control

In Tables 17-27 each figure on average bacteria count per birdrepresents the average of 5 birds.

EXAMPLE 5

The objective of this study was to determine the effect of bleachmicrobiocidal control (20 ppm Cl₂ equivalent) and of microbiocidalcontrol with 1,3-dibromo-5,5-dimethylhydantoin (DBDMH) on organoleptictaste evaluation of both breast and thigh meat. Formal trained tastepanel evaluation was conducted. The trial was conducted using 49-day oldbirds which were processed unchallenged with external sources ofbacteria and under sterile conditions.

A total of 120 birds were used in this study. Sixty of the birds servedas a control group. These were subjected to treatment in a chill tankcontaining Clorox® bleach at a 20 ppm Cl₂ equivalent level. The other 60birds were treated in a chill tank in the same fashion except that thechilling water contained DBDMH at the level of 20 ppm Cl₂ equivalent.During the 1.5 hour chilling period in the chill tank, the contents ofthe tank were vigorously stirred every 10 minutes. After the 1.5 hourchilling period, the whole birds were individually bagged and placed ina commercial refrigerator for 20 days of storage. After aging,individual breast and thigh samples were cut and cooked to an internaltemperature of 190° F. Taste evaluation was determined using 10 trainedtaste panel experts. A Ranking System (“1” or “2”) was used where “1”represents the better tasting sample. A simple average of subjectevaluations or rankings per person were used. Statistical evaluation wasemployed by using each subject as a block employed delta 0.05.

Tables 28 and 29 set forth the results of these taste evaluations. TABLE28 Effect of Chill Tank Water Treatment On Taste Preference (Breast MeatEvaluation) SUMMARY - Tasting Ranking¹ Water Treatment S1 S2 S3 S4 S5 S6S7 S8 S9 S10 Mean² None (20 ppm Cl₂ equivalent 2 1 1 1 2 1 1 2 1 2 1.4 ableach control) DBDMH (20 ppm Cl₂ equivalent) 1 2 2 2 1 2 2 1 2 1 1.6 a¹S(subject) = trained taste panelist subject number.²NOTE: Means within a row without a common superscript are significantlydifferent (P < 0.05) as determined by Least Significant Difference.

TABLE 29 Effect of Chill Tank Water Treatment On Taste Preference (ThighMeat Evaluation) SUMMARY - Tasting Ranking¹ Water Treatment S1 S2 S3 S4S5 S6 S7 S8 S9 S10 Mean² None (20 ppm Cl₂ 1 2 2 1 1 2 2 2 1 2 1.6 aequivalent bleach control) DBDMH (20 ppm Cl₂ 2 1 1 2 2 1 1 1 2 1 1.4 aequivalent)¹S(subject) = trained taste panelist subject number.²NOTE: Means within a row without a common superscript are significantlydifferent (P < 0.05) as determined by Least Significant Difference.

EXAMPLE 6

The objective of this study was to determine the effect of Clorox®bleach and 1,3-dibromo-5,5-dimethylhydantoin (DBDMH) on individualcarcass bacteria field strains after 1.5 hour in a chill tank solutionand spoilage 20-day shelf life longevity (caused by bacteriacontamination) in a Graded Level Study Model. After normal processing of56-day-old birds, carcasses were immersed first in a warm bathcontaining 10⁴ CFU's per mL Escherichia coli, 10⁴ CFU's per mLSalmonella enteritidis, 10⁴ CFU's per mL Pseudomonas aeruginosa, 10⁴CFU's per mL Campylobacter jejuni, and 10⁴ CFU's per mL spoilagebacteria each from two strains (Listeria monocytogenes and Shigellasonnei). Carcasses were then immersed in a chill tank “soup”, containingnormal organic fluids (blood, fat, skin, and meat particles) andcontaining various disinfectants (termed test materials). These testswere conducted at pH 8 (adjusted by trisodium phosphate). Skinpigmentation (Minolta Color Meter L value or Lightness, a value orredness and b value or yellowness) was determined before and afterprocessing. Post-chilling skin bacteria of various strains weredetermined over a 20-day period. Sensory evaluation was determined todemonstrate spoilage times and shelf-life. After salmonella infection inchill tanks, USDA HACCP salmonella detection was simulated and reported.

The materials tested and the experimental design of these test were assummarized in Table 30. TABLE 30 Test Group Test Material (Chill Tank)Reps Birds/Rep 1 None (Control) 10 12 2 Clorox ® bleach (20 ppm Cl₂ 1012 equivalent 3 DBDMH (5 ppm Cl₂ equivalent) 10 12 4 DBDMH (10 ppm Cl₂equivalent) 10 12 5 DBDMH (15 ppm Cl₂ equivalent) 10 12 6 DBDMH (20 ppmCl₂ equivalent) 10 12 7 DBDMH (25 ppm Cl₂ equivalent) 10 12

A DBDMH stock solution and DBDMH test solutions of the concentrationsspecified in Table 30, a bacteria stock solution, and a “chicken soup”were prepared as in Example 3. In addition, the bacterial brothtreatments, the whole bird wash sampling procedure, and themethodologies used for quantitative or qualitative determinations forbacterial organisms were conducted as in Example 3.

The trial events and experimental design used in this group of testswere the same as in Example 5 with the following exceptions:

-   a) The temperature during the 20-day period of storage in the    refrigerator was 4° F.-   b) Observations of the degree of “bloating” (defined as water or air    additions under the skin area considered objectionable) were    conducted on all processed birds.-   c) On each of sampling days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and    20, ten carcasses from each treatment were analyzed by removing 23.8    cm² of skin from the breast right up to the neck using a template    and a sterile scalpel. Each skin sample was placed in a bag with 15    mL Butterfield's Phosphate Buffer Solution (BPBS) added and treated    in a Stomacher bag for 60 seconds. A 10-fold dilution series of the    mixture was made in BPBS and two parallel samples of 20 mL each were    spread on the appropriate plate count agar for determination of the    total viable numbers. The plates were incubated at 35° C. for 24    hours. Mean values were calculated from the two determinations of    the three samples taken from each combination of chilling and    storage. Bacterial numbers were reported as pooled or averaged log₁₀    colony-forming units (CFU's) per square centimeter.-   d) Also on sampling day 0, 102 total of the remaining 110 carcasses    from each treatment (all bloating and oddly processed birds were    removed) were “whole bird” washed by the sampling procedure    described in Example 3. Salmonella detection were noted and reported    as number of positive salmonella colonies per 51 birds and % of    total.

Tables 31-34 summarize the results of this group of tests. TABLE 31Salmonella Positive Samples (Number per 51)¹ (Birds were inoculated withWater Treatment Salmonella prior to chilling) None (Control) 32/51(62.74%) Clorox ® Bleach (20 ppm) 11/51 (22.57%) DBDMH (5 ppm)  7/51(13.73%) DBDMH (10 ppm) 4/51 (7.84%) DBDMH (15 ppm) 2/51 (3.92%) DBDMH(20 ppm) 1/51 (1.96%) DBDMH (25 ppm) 0/51 (0.00%)¹Twelve (12) per 51 or less is considered to be statistically acceptableby USDA HACCP standards. A total of 102 birds were used to determinesalmonella positive samples and a simple average determined.

TABLE 32 Water Treatment Birds (Number per 60 birds processed)¹ None(Control) 1/120 (0.83%) Clorox ® Bleach (20 ppm) 0/120 (0.00%) DBDMH (5ppm) 2/120 (1.67%) DBDMH (10 ppm) 0/120 (0.00%) DBDMH (15 ppm) 1/120(0.83%) DBDMH (20 ppm) 0/120 (0.00%) DBDMH (25 ppm) 1/120 (0.83%)¹Four (4) or more per treatment is considered to be highlyobjectionable.

TABLE 33 Sensory Score (days post-processing)^(1,2) Water Treatment 5days 10 days 15 days 20 days None (Control) 5.6 c 7.3 c 8.2 c 9.0 dClorox ® bleach (20 ppm) 3.8 b 3.6 b 5.5 b 7.1 c DBDMH (5 ppm) 2.4 ab3.2 b 3.9 a 5.6 a DBDMH (10 ppm) 1.9 ab 2.3 a 3.4 a 4.8 a DBDMH (15 ppm)1.3 a 2.1 a 2.6 a 4.9 a DBDMH (20 ppm) 1.1 a 1.8 a 2.7 a 4.3 a DBDMH (25ppm) 1.4 a 2.1 a 2.3 a 4.6 a¹Continuous scale for non-structured fresh inside carcass odor sensoryattributes ranges from value 1.0 (the lowest intensity) to value 9.0(the highest intensity).NOTE:Means within a row without a common superscript are significantlydifferent (P < 0.05) as determined by Least Significant Difference.²Five (5) or more is considered to be highly objectionable.

TABLE 34 Skin Pigmentation¹ Mean Pre-Chill Mean Post-Chill Water MinoltaValue² Minolta Value² Treatment L a B L a b None (Control) 59.72 a 4.34a 13.67 a 51.84 a 5.12 a 15.27 a Clorox ® 60.76 a 4.93 a 13.74 a 55.81 a5.08 a 15.49 a Bleach (20 ppm) DBDMH 58.80 a 4.67 a 13.61 a 52.68 a 5.42a 15.64 a (5 ppm) DBDMH 59.97 a 4.31 a 13.64 a 53.19 a 5.69 a 15.75 a(10 ppm) DBDMH 58.43 a 4.84 a 13.81 a 54.21 a 5.55 a 15.64 a (15 ppm)DBDMH 58.54 a 4.99 a 13.67 a 53.74 a 5.49 a 15.80 a (20 ppm) DBDMH 58.97a 4.68 a 13.50 a 54.25 a 5.63 a 15.76 a (25 ppm)¹NOTE:Means within a row without a common superscript are significantlydifferent (P < 0.05) as determined by Least Significant Difference.²Skin pigmentation (Minolta Color Meter L value or Lightness, a value orredness and b value or yellowness).

Results from the above tests on the effect of chill tank treatment ongrowth of Pseudomonas species on the chicken skin are graphicallydepicted in FIG. 1. FIG. 2 depicts graphically the results of the abovetests on the effect of chill tank treatment on growth of total aerobicbacteria on the chicken skin.

EXAMPLE 7

A study was carried out to determine the effectiveness of severalmicrobiocidal compounds of this invention, as well as sodiumhypochlorite when used as carcass rinses. The microbiocides of thisinvention used in this study were 1,3-dibromo-5,5-dimethylhydantoin(DBDMH), N,N′-bromochloro-5,5-dimethylhydantoin (BCDMH) and Stabrom® 909biocide (Albemarle Corporation), a concentrated alkaline aqueoussolution produced from bromine chloride and sulfamate anion (SSBC).

After normal processing of 56-day-old birds, carcasses were immersedfirst in a warm bath containing 10⁴ per mL Escherichia coli, 10⁴ per mLSalmonella enteritidis, 10⁴ per mL Pseudomonas aeruginosa, 10⁴ per mLCampylobacter jejuni, and 10⁴ per mL spoilage bacteria each from twostrains (Listeria monocytogenes and Shigella sonnei). Carcasses werethen immersed in a chill tank “soup”, containing normal organic fluids(blood, fat, skin, and meat particles) and containing variousdisinfectants (termed test materials). These whole bird bacteria counttests were conducted at pH 8. The effect of the test compounds on skinpigmentation was determined by use of Minolta Color Meter L value orLightness, a value or redness and b value or yellowness. Post-chillingskin bacteria of various strains were determined over a 20-day period.Spoilage, using sensory odors as a model, determined time required tocreate a putrid/ammonia-like odor. After salmonella infection in chilltanks, USDA HACCP salmonella detection was simulated and reported. Table35 describes the test material dosages and overall design of this groupof tests. TABLE 35 Test Group Test Material (Chill Tank) Reps Birds/Rep1 None (Control) 10 12 2 Clorox ® bleach (20 ppm Cl₂ 10 12 equivalent)during chilling 3 DBDMH (20 ppm Cl₂ equivalent) 10 12 during chilling 4BCDMH (20 ppm Cl₂ equivalent) 10 12 during chilling 5 SSBC carcass spray(3% liquid 1 10 pre-chill application)

DBDMH and BCDMH stock solutions and diluted test solutions (20 ppm Cl₂equivalent), a bacteria stock solution, and a “chicken soup” wereprepared as in Example 3 except that the Stabrom® 909 biocideconcentrate was diluted by adding 30 mL per liter of water just prior toapplication. This diluted solution was sprayed on the birds, both insideand outside, in quantities of 200 mL per bird. In addition, thebacterial broth treatments, the whole bird wash sampling procedure, andthe methodologies used for quantitative or qualitative determinationsfor bacterial organisms were conducted as in Example 3.

The details concerning the trial events used as well as the detailedexperimental design used in these tests were the same as described inExample 6. The only exceptions were:

-   a) In the case of the birds of Test Group 5 (note Table 35), while    the carcass was still warm, the 10 birds were each sprayed both    internally and externally, using a misting hand-held nozzle, with    200 mL of the 3% solution of Stabrom 909 biocide (SSBC). Previous    quality control trials using dye had ensured that complete carcass    coverage was achieved with the use of 200 mL of liquid spray. The    spray was allowed to stay on the warm carcasses for 60 seconds.-   b) The treatment on sampling day 0 of 102 total of the remaining 110    carcasses from each treatment involving “whole bird” washing and    Salmonella detection, all as described in Example 6, was applied    only to the birds of Test Groups 1-4 (note Table 35).

Tables 36-39 summarize the results of this group of tests. The effect ofthe chill tank treatment of this Example on growth of Pseudomonasspecies on chicken skin are graphically depicted in FIG. 3. FIG. 4depicts graphically the results of the tests of this Example on theeffect of chill tank treatment on growth of total aerobic bacteria onthe chicken skin. TABLE 36 Salmonella positive samples (Number 51)¹(Birds were inoculated Water Treatment with Salmonella prior tochilling) None (Control) 21/51 (41.18%) Clorox ® bleach (20 ppm)  8/51(15.68%) DBDMH (20 ppm Cl₂ equivalent) 1/51 (1.96%) during chillingBCDMH (20 ppm Cl₂ equivalent)  6/51 (11.76%) during chilling¹Twelve (12) per 51 or less is considered to be statistically acceptableby USDA HACCP standards. A total of 102 birds were used to determinesalmonella positive samples and a simple average determined.

TABLE 37 Bloating (Number per Water Treatment 60 birds processed)¹ None(Control) 0/120 (0.00%) Clorox ® bleach (20 ppm) 0/120 (0.00%) DBDMH (20ppm Cl₂ equivalent) 0/120 (0.00%) during chilling BCDMH (20 ppm Cl₂equivalent) 0/120 (0.00%) during chilling¹Four (4) or more per treatment is considered to be highlyobjectionable.

TABLE 38 Sensory Score (days post-processing)^(1,2) Water Treatment 5days 10 days 15 days 20 days None (Control) 2.4 b 4.8 c 6.9 c 9.0 dClorox ® 1.3 ab 2.4 b 4.6 b 6.8 c bleach (20 ppm) DBDMH (20 ppm Cl₂ 0.6a 1.2 a 3.2 a 3.4 a equivalent) during chilling BCDMH (20 ppm Cl₂ 1.4 ab1.8 ab 2.7 a 4.8 b equivalent) during chilling¹Continuous scale for non-structured fresh inside carcass odor sensoryattributes ranges from value 1.0 (the lowest intensity) to value 9.0(the highest intensity). NOTE: Means within a row without a commonsuperscript are significantly different (P < 0.05) as determined byLeast Significant Difference.²Five (5) or more is considered to be highly objectionable.

TABLE 39 Skin Pigmentation¹ Mean Pre-Chill Mean Post-Chill MinoltaValue² Minolta Value² Water Treatment L a b L a b None (Control) 52.61 a3.25 a 11.43 a 47.21 a 4.24 a 12.44 a Clorox ® 52.76 a 3.32 a 11.84 a47.43 a 4.85 a 12.67 a bleach (20 ppm) DBDMH (20 ppm 52.23 a 3.13 a11.63 a 48.02 a 4.69 a 12.47 a Cl₂ equivalent) during chilling BCDMH (20ppm 52.11 a 3.82 a 11.26 a 46.93 a 4.44 a 12.60 a Cl₂ equivalent) duringchilling SSBC Carcass 52.61 a 3.67 a 11.15 a 47.03 a 4.51 a 12.55 aSpray (3% liquid pre-chill application)¹NOTE: Means within a row without a common superscript are significantlydifferent (P < 0.05) as determined by Least Significant Difference.²Skin pigmentation (Minolta Color Meter L value or Lightness, a value orredness and b value or yellowness)³All treatment skin pigmentation were measured on 120 birds, except forSSBC where only 10 birds were employed.

A number of tests have been carried out demonstrating the microbiocidaleffectiveness of several microbiocides in eradicating or controllingvarious bacteria species of the types present in poultry processingsystems.

One such series of tests involved determinations of microbiologicalcontrol against Escherichia coli bacteria. Another set of tests involveddeterminations of microbiological control against Enterococcus faecium.In each case, comparative tests were carried out in the same mannerutilizing the AOAC test method. Such test involves exposing a culture ofthe microorganism to various concentrations of a test solution preparedfrom an aqueous stock solution of the compound under test. At varioustime intervals the halogen in the test suspensions is chemicallyneutralized, and the amount of viable bacteria remaining is enumeratedby plating onto nutrient agar and incubating for 2 days at 37° C.Results are expressed at the log₁₀ colony forming units (CFU). Theconcentration of the compound required to achieve complete kill (i.e.,no viable bacteria remain) within 30 seconds is determined in the test.

Table 40 summarizes the data obtained in the tests using respectively,1,3-dibromo-5,5-dimethylhydantoin (DBDMH) andN,N-bromochloro-5,5-dimethylhydantoin (BCDMH) and in which themicroorganism in each case was Escherichia coli. It can be seen that1,3-dibromo-5,5-dimethylhydantoin passed the test at one milligram ofbromine, as Br₂, per liter of water, as evidenced by the complete killwithin 30 seconds, whereas 1,3-bromochloro-5,5-dimethylhydantoinrequired two milligrams of bromine, as Br₂, per liter of water toachieve complete kill within 30 seconds. TABLE 40 EFFECTIVENESS AGAINSTESCHERICHIA COLI Log₁₀ CFU Log₁₀ CFU Concentration Recovered Recoveredmg/L as Br₂ Contact Time Using DBDMH Using BCDMH 0.5 mg/L 30sec   >4.48 >4.48 1 min 1.70 4.46 2 min 0 1.65 3 min 0 0 4 min 0 0 5 min0 0 10 min  0 0 1.0 mg/L 30 sec   0 >4.48 1 min 0 0.7 2 min 0 0 3 min 00 4 min 0 0 5 min 0 0 10 min  0 0 2.0 mg/L 30 sec   0 0 1 min 0 0 2 min0 0 3 min 0 0 4 min 0 0 5 min 0 0 10 min  0 0

Table 41 summarizes the data obtained in the tests using respectively1,3-dibromo-5,5-dimethylhydantoin (DBDMH) andN,N′-bromochloro-5,5-dimethylhydantoin (BCDMH) and in which themicroorganism in each case was Enterococcus faecium. Table 44 shows that1,3-dibromo-5,5-dimethylhydantoin passed the test at one milligram ofbromine, as Br₂, per liter of water, as evidenced by the complete killwithin 30 seconds, whereas N,N′-bromochloro-5,5-dimethylhydantoinrequired two milligrams of bromine, as Br₂, per liter of water toachieve complete kill within 30 seconds. TABLE 41 EFFECTIVENESS AGAINSTENTEROCOCCUS FAECIUM Log₁₀ CFU Log₁₀ CFU Concentration RecoveredRecovered mg/L as Br₂ Contact Time Using DBDMH Using BCDMH 0.5 mg/L 30sec   4.32 >4.48 1 min 2.36 3.53 2 min 0.00 2.63 3 min 0.00 0.00 4 min0.00 0.00 5 min 0.00 0.00 10 min  0.00 0.00 1.0 mg/L 30 sec   0.00 >4.481 min 0.00 2.38 2 min 0.00 0.00 3 min 0.00 0.00 4 min 0.00 0.00 5 min0.00 0.00 10 min  0.00 0.00 2.0 mg/L 30 sec   0.00 0.00 1 min 0.00 0.002 min 0.00 0.00 3 min 0.00 0.00 4 min 0.00 0.00 5 min 0.00 0.00 10 min 0.00 0.00

Table 42 summarizes test results performed at MBEC Biofilm Technologies,Inc., Calgary, Canada on the effectiveness of various biocides onbiofilm removal. The test procedure, developed at the University ofCalgary, utilizes a device which allows the growth of 96 identicalbiofilms under carefully controlled conditions. The device consists of atwo-part vessel comprised of an upper plate containing 96 pegs thatseals against a bottom plate. The bottom plate can consist of either atrough (for biofilm growth) or a standard 96-well plate (for biocidechallenge). The biofilms develop on the 96 pegs. The device has beenused as a general method for evaluating the efficacy of antibiotics andbiocides towards biofilms. See in this connection H. Ceri, et al., “TheMBEC Test: A New In Vitro Assay Allowing Rapid Screening for AntibioticSensitivity of Biofilm”, Proceedings of the ASM, 1998, 89, 525; Ceri, etal., “Antifungal and Biocide Susceptibility testing of Candida Biofilmsusing the MBEC Device”, Proceedings of the Interscience Conference onAntimicrobial Agents and Chemotherapy, 1998, 38, 495; and H. Ceri, etal., “The Calgary Biofilm Device: A New Technology for the RapidDetermination of Antibiotic Susceptibility of Bacterial Biofilms”,Journal of Clinical Microbiology, 1999, 37, 1771-1776.

Six biocide systems were evaluated using the above test procedure andtest equipment. Five of these systems were oxidizing biocides, viz.,chlorine (from NaOCl), halogen (from NaOCl+NaBr), halogen (from BCDMH),bromine (from DBDMH), and chlorine (from trichloroisocyanuric acid), allexpressed as bromine as Br₂ in mg/L, so that all test results wereplaced on the same basis. The sixth biocide was glutaraldehyde, anon-oxidizing biocide.

These biocide systems were used to challenge biofilms of Pseudomonasaeruginosa (ATCC 15442). This is a Gram (−) bacterium which isubiquitous in microbiological slimes found in many water systems. See inthis connection J. W. Costerton and H. Anwar, “Pseudomonas aeruginosa:The Microbe and Pathogen”, in Pseudomonas aeruginosa Infections andTreatment, A. L. Baltch and R. P. Smith editors, Marcel Dekkerpublishers, New York, 1994. In the field of poultry processing, S.Notermans, J. Dormans, and G. C. Mead, Biofouling, 1991, Vol. 5, pages21-36, report observation of biofilms in poultry slaughter houses by useof scanning electron microscopy.

In Table 42 the MBEC (minimum biofilm eradication concentration) resultspresented are for the one-hour biocide contact time used in the test.The values given for the halogen containing biocides are expressed interms of mg/L of bromine as Br₂. The data on the glutaraldehyde is interms of mg/L as active ingredient. The data indicate that the DBDMH wasmore effective than any of the other biocides tested under theseconditions with an MBEC of 1.4 mg/L of bromine, as Br₂. In fact, onlyslightly more than one-half as much bromine from DBDMH was required toremove the biofilm as compared to the total halogen, expressed as Br₂,that was required from BCDMH. TABLE 42 EFFECTIVENESS AGAINST PSEUDOMONASAERUGINOSA BIOFILM Biocide System MBEC MBEC, avg. Chlorine (from NaOCl)5.0, 2.5 3.8 Halogen (from NaOCl + NaBr) 2.5, 2.5 2.5 Halogen (fromBCDMH) 2.5, 2.5 2.5 Bromine (from DBDMH) 1.4, 1.4 1.4 Chlorine (fromTrichloroisocyanuric acid) 2.6, 1.3 2.0 Glutaraldehyde 50, 50 50

In another group of tests, the results of which are depicted in FIGS. 5and 6 bromine-based microbiocides of this invention were utilized intests illustrating their effectiveness in eradicating or controllingHeterotrophic Plate Count bacteria i.e., a mixture ofnaturally-occurring pathogenic bacteria of various unidentified species.These bacteria were challenged both in the form of biofilms and inplanktonic form.

The experimental conditions utilized in these tests involved use of anapparatus consisting of three parallel transparent PVC sampling pipes.These pipes were used for collection of biofilm (i.e., sessile orsurface attached) bacteria samples; one as control pipe, one for arelatively low biocide concentration and the third for a higher biocideconcentration. The biocide challenge in each case was divided into threephases. First was a 14-day inoculation. Next was a 48-hour disinfectionperiod. Finally a 2-week recovery period was provided. The biocide undertest was slug-dosed and during the first hour of exposure, theconcentration was adjusted to achieve the desired concentration level.

The source of the naturally-grown heterotrophic plate count (HPC)bacteria was sediment and associated water collected from therecirculating hot water system of a hospital. Filter cartridges wereinserted into the hospital water system and after about two months asuitable amount of sediment had accumulated on the filters. Thecollected filter/water suspension was then harvested for culturing. Theinoculum for the biocide challenge experiments consisted ofdechlorinated tap water, HPC-cultured stock solution, and a nutrientsupplement solution. The inoculum was incubated at 37° C. for 14-daysprior to the start of the test. The inoculum along with additionaldechlorinated tap water was introduced into the apparatus composed ofthe three parallel transparent PVC sampling pipes. This mixture wasrecirculated throughout the apparatus intermittently at the rate of 3.2gallons per minute for 14-days to produce a consistent biofilm andplanktonic HPC bacteria population.

Samples of these bacteria were collected at the end of the 14-dayinoculation period before the biocide challenge. In each test, the HPCbacteria was then challenged with a specified level of a bromine-basedbiocide, and samples were taken at 1, 2, 3, 12, and 48-hour intervals.These samples were taken by swabbing the inner surface of a premeasuredsection (length, 17/32 inch) of the transparent PVC sampling pipe. Theswabs were vortexed for 1 minute in 5 mL of deionized water with 0.1 mLof a neutralizer (to remove residual bromine) before plating.Concurrently, water samples were taken for enumeration of the planktonicHPC bacteria.

After the 48-hour biocide challenge period, the procedure involvedproviding the 2-week recovery period. The purpose of providing thisrecovery period was to determine how quickly the viable HPC bacteriathat were still present repopulated both the biofilm and, in planktonicform, the recirculating water. Thus, the recirculating water was drainedfrom the test apparatus and the apparatus was refilled withheat-sterilized tap water which was also allowed to recirculateintermittently as before. After 7 and 14 days the apparatus wasresampled and biofilm and planktonic HPC bacteria were enumerated in thesame manner as done previously.

The results of these test are presented in graphical form in the FIGS. 5and 6. The test results depicted in FIG. 5 involved use of1,3-dibromo-5,5-dimethylhydantoin (Albrom™ 100 biocide, AlbemarleCorporation) as the source of active bromine species. This microbiocidewas used in these tests at levels of 0.5 ppm and 5 ppm as bromine tochallenge biofilm-associated HPC bacteria. Also, a control was carriedout in the same manner except that no biocide was applied. It can beseen from FIG. 5 that at the higher bromine concentration, within twelvehours almost 99.9% of the HPC bacteria were eradicated. At 0.5 ppm asbromine, over 99% of the HPC bacteria were eradicated within threehours. It can also be seen that within the 48-hour biocide challengeperiod, the very small amounts of the viable HPC biofilm that stillremained were beginning to recover in both tests in which the brominebiocide was used. These test results also indicate that for the HPCbacteria to reestablish populations close to their original levels, arecovery period of substantially greater than two weeks would have beenrequired.

In the tests of FIG. 6 the active bromine species used and theirconcentrations were the same as in FIG. 5, and a control was used.However, in these tests the HPC bacteria were in planktonic form. It canbe seen that at the higher bromine concentration, almost 99.99% of theplanktonic HPC bacteria were eradicated within twelve hours. At 0.5 ppmas bromine and within three hours, almost 99% of the planktonic HPCbacteria were eradicated. It can also be seen that within the 48-hourbiocide challenge period, the very small amounts of the viableplanktonic HPC bacteria that still remained were beginning to recover inboth tests in which the bromine biocide was used. These test resultsalso indicate that for the planktonic HPC bacteria to reestablishpopulations close to their original levels, a recovery period of morethan two weeks would have been required.

In the practice of this invention, combinations of different sanitizingsteps using different microbiocidal agents, at least one of which is amicrobiocide of this invention, preferably one or more bromine-basedmicrobiocidal agents of this invention, can prove useful. For example, amicrobiocide of this invention, preferably a bromine-based microbiocideof this invention, can be applied to or contacted with various surfacesassociated with the poultry processing such as conduits, tanks (e.g.,the scalding tank(s), chill tank(s), conveyor belts or conveyor lines,and the poultry carcasses themselves can be treated with anantimicrobial agent such as solutions or gels containing carboxylicacids (e.g., acetic or lactic acid) and/or peroxycarboxylic acids, suchas peracetic acid, peroxyoctanoic acid, peroxydecanoic acid, or thelike. Use of such carboxylic acids is described for example in U.S. Pat.No. 6,113,963. The result of such combined operations is highlyeffective sanitization. In fact, it is contemplated that thiscombination of operations will result in a greater extent ofmicrobiological eradication than has been generally achievableheretofore, especially when the bromine-based biocide used is1,3-dibromo-5,5-dimethylhydantoin and the carboxylic acid used isperacetic acid. Indeed the combined effect of these microbiocides may besynergistic.

Another microbiocide which can be utilized in combined operationspursuant to this invention is trisodium phosphate, a material whichaccording to Capita et al., Meat Science, 2000, 55 (4), 471-474, hasbeen approved by the USDA as an aid to eliminate Salmonella on rawpoultry carcasses. In the combined operations trisodium phosphate isapplied to the poultry carcasses, and one or more of the microbiocidesof this invention, preferably one or more of the bromine-basedmicrobiocides of this invention, are utilized in sanitizing theequipment, instruments, and/or apparatus associated with the processingof the poultry. Also pursuant to this invention the combined operationscan utilize chlorine dioxide treatments along with use of themicrobiocides of this invention. Smith, Meat Processing, 1996, 35(10),47 indicates that chlorine dioxide had been approved by the US FDA foruse in poultry processing water, and in the practice of this inventionone or more microbiocides of this invention, preferably one or more ofthe bromine-based microbiocides of this invention, are utilized insanitation of various items of equipment, instruments, and/or apparatusutilized in the processing of the poultry, and chlorine dioxide is usedto sanitize at least some of the poultry processing water.

Another way by which combined operations pursuant to this invention canbe carried out involves administering to the digestive tract of thepoultry a suitable biological pathogen-control agent, such as byincluding such biological agent in the drinking water for the fowl, oron or in the feed for the fowl. Illustrative biological pathogen-controlagents which may be used in this manner include certain strains of E.coli described in U.S. Pat. No. 6,083,500. Thus in the practice of thisinvention, such a biological pathogen-control agent is provided to thefowl for consumption by drinking and/or eating, and amicrobiocidally-effective amount of an aqueous solution of at least onemicrobiocide of this invention, which preferably is at least onebromine-based microbiocide of this invention, is used in disinfecting orsanitizing equipment, instruments, apparatus, and/or water used in theprocessing of poultry, and/or of carcasses and/or parts of poultryresulting from the processing of poultry.

Still another combined operation involves (i) treating the carcasses ofthe fowl with immobilized lactoferrin antimicrobial agents as describedin U.S. Pat. No. 6,172,040 B1 and (ii) disinfecting or sanitizing all ora portion of the equipment, instruments, apparatus, and/or water used inthe processing of poultry by contacting the same with amicrobiocidally-effective amount of an aqueous solution of at least onemicrobiocide of this invention, which preferably is at least onebromine-based microbiocide of this invention.

Automated dispensing equipment suitable for use in dispensing themicrobiocides of this invention has been described in the literature andto at least some extent is available in the marketplace. For a referenceto such equipment, see for example U.S. Pat. No. 5,683,724 wherein anautomated dispensing system is described.

While chemists understand what is meant by “aqueous” in connection witha solution or medium or the like, it is probably desirable to state justwhat “aqueous” means. The adjective “aqueous” means that the solution ormedium or whatever other noun the adjective modifies, can be waterwhether highly purified or of ordinary purity such as emanates from thefaucet. Since we are dealing with processing of food, it stands toreason that one would not use sewer water or water containing lethaldoses of poisons such as cyanide. Besides naturally-occurring traceimpurities that may be present in, say, potable water in general, suchas ordinary well water or municipal water, the adjective “aqueous” alsopermits the presence in the water of dissolved salts that are formed inthe course of forming a bromine-based microbiocide in the water, e.g.,by reaction between bromine chloride and sodium sulfamate in anoverbased aqueous solution. In addition, “aqueous” permits the presenceof small amounts of innocuous non-harmful, water-soluble organicsolvents such as ethyl alcohol which can be used as a solvent for the1,3-dihalo-5,5-dialkylhydantoin(s). Also “aqueous” permits the presencein the water of the amount of the halogen-based microbiocide itself tothe extent that it may dissolve in the water, plus any dissolvedreactant(s) that may remain after the reaction. Also the water maycontain a few atoms that may dissolve from the vessel in which thereaction takes place, plus air-borne impurities that may find their wayinto the water. The point here is that the term “aqueous” does notrestrict the medium or solvent to absolutely pure water—the aqueoussolution or medium or the like can contain what would normally bepresent and/or reasonably be expected to be present in it under theparticular circumstances involved when employing ordinary common sense.

Compounds referred to by chemical name or formula anywhere in thisdocument, whether referred to in the singular or plural, are identifiedas they exist prior to coming into contact with another substancereferred to by chemical name or chemical type (e.g., another component,a solvent, or etc.). It matters not what chemical changes, if any, takeplace in the resulting mixture or solution, as such changes are thenatural result of bringing the specified substances together under theconditions called for pursuant to this disclosure. As an example, thephase “solution of at least one 1,3-dihalo-5,5-dialkylhydantoin” andphrases of similar import signify that just before being brought intocontact with an aqueous medium such as water, the at least one1,3-dihalo-5,5-dialkylhydantoin referred to was the specified1,3-dihalo-5,5-dialkylhydantoin. The phrase thus is a simple, clear wayof referring to the solution, and it is not intended to suggest or implythat the chemical exists unchanged in the water. The transformationsthat take place are the natural result of bringing these substancestogether, and thus need no further elaboration.

Also, even though the claims may refer to substances in the presenttense (e.g., “comprises”, “is”, etc.), the reference is to the substanceas it exists at the time just before it is first contacted, blended ormixed with one or more other substances in accordance with the presentdisclosure.

Except as may be expressly otherwise indicated, the article “a” or “an”if and as used herein is not intended to limit, and should not beconstrued as limiting, the description or a claim to a single element towhich the article refers. Rather, the article “a” or “an” if and as usedherein is intended to cover one or more such elements, unless the textexpressly indicates otherwise.

All documents referred to herein are incorporated herein by reference intoto as if fully set forth in this document.

This invention is susceptible to considerable variation within thespirit and scope of the appended claims.

1. Apparatus which comprises at least one poultry chill tank containingan aqueous medium into which a plurality of poultry carcasses areimmersed and wherein an effective microbial inhibiting amount of activebromine is present in said medium, said amount of active bromineresulting from the addition to water before it enters said tank or whileit is in said tank, or both, of (i) at least one1,3-dibromo-5,5-dialkylhydantoin in which one of the alkyl groups is amethyl group and the other alkyl group contains in the range of 1 toabout 4 carbon atoms or (ii) a solution thereof, or (iii) both of (i)and (ii), so that contamination of immersed carcasses by microorganismsis inhibited.
 2. Apparatus as in claim 1 wherein said at least one1,3-dibromo-5,5-dialkylhydantoin is 1,3-dibromo-5,5-dimethylhydantoin.3. Apparatus as in each of claims 1 or 2 wherein said effectivemicrobial inhibiting amount of active bromine in said medium is up toabout 100 ppm (wt/wt).
 4. Apparatus as in each of claims 1 or 2 whereinthe aqueous medium in said tank is at a temperature of up to about 4.5°C.